PROPOSAL

A. Basic Information

1. Project title: In vitro Regeneration through Somatic

Embryogenesis of Kawayan Kiling
(Bambusa vulgaris Schrad ex. Wendl)
and Bolo [Gigantochloa levis (Blanco)
Merr.]

2. Project Leader: Janine F. Cortiguerra/Female

Project Staff: Kevin Philip M. Olaya
Laborer (1)

3. Implementing Agency: Ecosystems Research & Development

Bureau

4. Place of Implementation: ERDB Tissue Culture Laboratory

College, Laguna

5. Funding Agency: Ecosystems Research & Development

Bureau

6. Duration: Three years

7. Total Budget Cost: P 1.212 M

B. Technical Description

Rationale

Bamboo is considered one of the most important non-timber forest

products in the Philippines. It plays a vital role in the world’s industrial and
domestic economies. It is also considered as wood substitute due to its
rapid growth, availability, desirable properties and plentiful uses. Bamboo
is utilized for construction of low-cost housing, for reforestation, for
landscaping purposes, for pulp and paper manufacture, handicrafts and for
cottage industries. It also supports other industries like housing, fishing,
banana and the food industry.

New industrial products out of bamboo are also being developed.

The production of charcoal and light distillates from bamboo is developed
by carbonization. Other products include the powder of bamboo leaf
extract, bamboo leaf flavonoids, beer fortifying bamboo leaf extract and
bamboo leaf beverage (Virtucio et al., 1999).

In 2015, the global market for bamboo rose to US$17 billion from
US$7 billion in 2009 based on figures from the Philippine Bamboo Industry
Cluster Report. With this growing market, the Philippines generated
P306.3 million in investments, P261.8 million worth of sales, and created
13,103 jobs from 2012-2014. The country was ranked as the 5th largest
bamboo exporter in the world in 2010.

To fully harness bamboo’s immense potential, the Department of

Environment and Natural Resources (DENR) in partnership with the
Climate Change Commission and other government agencies and
stakeholders aim to ensure the success of promoting bamboo as an
effective tool to mitigate adverse effect of climate change and provide
income to the poor. DENR plans to establish one million hectares of
bamboo plantation in critical areas and other sites covered by Enhanced
National Greening Program (ENGP) within the next six years (DENR,
2016).

Bambusa vulgaris Schrad ex. Wendl, also known as Kawayan

Kiling is the most commonly encountered cultivated bamboo which is
found in villages and riverbanks. This species is the most used of all
bamboos for making boats, boating poles, fencing and props (Roxas,
2012). Like other bamboo species, kawayan kiling is also known to be
monocarpic. It flowers and set seeds once and then die. Therefore,
propagation by seeds is difficult and impossible due to lack of flowering.
Vegetative propagation by rhizome, culm and branch cuttings is generally
used method for Kawayan kiling.

Gigantochloa levis (Blanco) Merr. or Bolo is also considered as one

of the economically important bamboo species in the Philippines. It is
generally found along creeks and riverbanks. The culms of Bolo are used
as props in the banana industries, fishing, furniture, handicraft making, and
building construction. Young shoots are also edible and nutritious. The
species can be propagated by culm, branch cutting or rhizome (Gonzales
et al., 1992).

With the large requirement for planting materials of bamboo,

harvests from conventional method are still insufficient. Tissue culture
offers an alternative method for rapid multiplication of bamboo species on
a large scale. This method uses planting materials or explants from elite
plants which later produce a number of true-to-type, virus-free and high
quality planting materials.

Somatic embryogenesis is one of the most powerful tools in plant

biotechnology. In vitro propagation through somatic embryogenesis allows
rapid multiplication of species. It is widely used to produce plants
commercially.

Development of an efficient micropropagation protocol capable of

generating large number of plants in short time and helps in multiplication
of high quality plants and conserving germplasm is necessary to
supplement and satisfy the demand.
 Objectives

General: To develop an efficient, reliable and cost effective in vitro

regeneration protocol using somatic embryogenesis of
Kawayan kiling (Bambusa vulgaris Schrad ex. Wendl) and
Bolo [Gigantochloa levis (Blanco) Merr.]

Specific:

1. To induce somatic embryogenesis on kawayan kiling and bolo.

2. To determine most suitable sterilization procedure for explants
establishment of kawayan kiling and bolo.
3. To determine most appropriate culture media/hormonal
combination for explant establishment and callus/somatic embryo
induction for mass propagation of kawayan kiling and bolo.
4. To determine the cost of producing bamboo plantlets through
somatic embryogenesis.

Review of Related Literature

A number of successful reports documenting propagation of bamboos

through in vitro techniques have been already published. Micropropagation is
a valuable method for rapid multiplication of difficult-to-propagate plants, both
for commercial production and germplasm conservation. In most studies
conducted, plants regenerated from shoot tips and nodal cuttings are
genetically stable and free from somaclonal variations. The level and kind of
plant growth regulators (PGRs) is necessary and largely determine the
success of tissue culture protocol. Incorporation of BAP and Kinetin into the
medium enhanced the axillary bud proliferation. IBA alone or in combination
with NAA are the most commonly used growth regulators for rooting bamboos
(Singh et al., 2012).

Nodal segments from adult plants were used to develop a method of in

vitro regeneration of Bambusa vulgaris. After 16 days, optimal shoot growth
was obtained on modified Murashige and Skoog (MMS) medium
supplemented with 2 mg/l BAP. Elongated shoots of Bambusa vulgaris rooted
(45-85%) when cultured in MMS with 20 mg/l IBA. The rooted and
acclimatized shoots were successfully transferred into the the field with 100%
plantlets survival (Ndiaye et al., 2006).

Phisohex-treated explants yielded the highest number of callus formed

in kawayan tinik (Bambusa blumeana Schultes f.) compared to those pre-
treated with citric/ascorbic acid and alcohol. Among the four (4) media
evaluated for kawayan tinik explants namely; Murashige and Skoog’s (MS),
Woody Plant Medium (WPM), N6 and B5, MS was the best media for callus
formation. Although browning was significantly higher in MS, it produced the
most number of profuse callus (Pollisco, 1991).
 Conventional micropropagation is a time consuming and labour
intensive process. In comparison with conventional clonal propagation and
other regeneration systems, somatic embryogenesis has powerful advantages
for mass multiplication. Micropropagation through somatic embryogenesis
offers not only a means for mass multiplication for biomass energy production,
but also provides the basis for the conservation of important elite and rare
plants that are threatened with extinction. The most commercially attractive
application of in vitro somatic embryogenesis is the high volume multiplication
of embryonic propagules (Jain et al., 2000).

According to Anis and Ahmad in 2016, somatic embryogenesis was

reported for the first time in 1982 in Bambusa arundinacea. Thereafter,
successful somatic embryogenesis has been reported in many species of
bamboos. However, in almost all cases, work has been limited to somatic
embryogenesis from either seeds or seedlings. The use of seeds or seedlings
for induction of somatic embryogenesis has some disadvantages. The
unknown genetic background of seeds and their highly variable nature are the
major drawbacks which discourage their use.

In 1985, Rao et al. were able to induce somatic embryogenesis in

Dendrocalamus strictus by culturing seeds (caryopsis) on basal B5 medium
supplemented with 2,4-D. When transferred to germination medium
containing IBA and NAA, about 40% of the somatic embryos developed into
plantlets.

Included in the study of Sood et al., in 2013 are different readings

regarding induction of somatic embryogenesis from different explants. Some
of these are from leaves in Bambusa multiplex (Jullien and Van, 1994),
anthers in Sinocalamus latiflorus (Tsayet al., 1990), roots of Bambusa
beecheyana Munro var. beecheyana (Chang and Lan, 1995), nodal segments
in Bambusa vulgaris (Rout and Das, 1997; Gielis, 1999) and Bambusa
ventricosa (Gielis, 1999) and inflorescences of Bambusa oldhamii Munro (Yeh
and Chang, 1986a), Bambusa beecheyana Munro var. beecheyana (Yeh and
Chang, 1986b) and Bambusa edulis (Lin et al., 2004a). However, in all these
cases regeneration of somatic embryos was either not achieved or it occurred
at too low a frequency to be used on a commercial scale.

In the study of Malini and Anandakumar in 2013, nodal segments from

field grown culture were also used. Optimum bud spread development was
obtained after 25-28 days cultivation to Murashige and Skoog (MS) medium
supplemented with 2.5 mg/l BAP and 2.5 mg/l Kin. MS with 7.5 mg/l IBA was
also proven the most suitable media for rooting of shoots. The in vitro
regenerated plantlets showed 80% survival when transferred to the field.

Arya et al. (2008) achieved successful somatic embryogenesis in D.

asper from nodal tissues and basal part of leaves. Callus cultures obtained on
2,4-D (30.0 μM) became highly embryogenic when transferred to medium
supplemented with 2,4-D (9.0 μM), BAP (0.88 μM) and IAA (2.85 μM).
Somatic embryos thus obtained, developed into plantlets within 30 days with
conversion rate of 70%.
 In the Philippines, somatic embryogenesis was induced on soft
internode tissue from ground corm of Dendrocalamus latiflorus cv. Machiku
(Zamora et al., 1988). Zamora and Gruezo (1990) also reported somatic
embryogenesis from excised embryos and seeds of Dendrocalamus strictus
inoculated on B5 media with 10 ppm 2,4-D. Eighty-five percent (85%) of the
embryogenic calli formed plantlets on ½ strength MS medium.

Methodology

Planting materials (culm cuttings) of kawayan kiling and bolo will be

purchased, thus explants for the laboratory trials will be readily available.
Additional planting materials will also be collected from the Bambusetum
located at Los Baños Experiment Station (LBES) which is under the
jurisdiction of Laboratory and Experimental Services Division (LESD).

A. Sterilization:
1. Nodal segments (4-5cm) will be collected from shoots/branches
of the mother plant of kawayan killing and bolo.
2. Leaf sheaths enclosing these nodes will be removed carefully.
3. Nodal segments will be washed with liquid soap and tap water
twice and will be subjected to the following disinfectants for
establishment of aseptic cultures:

 Fungicide – 0.5 gm/500ml water at 24 hours of soaking

 95% ethyl alcohol at 1 minute soaking with gentle agitation to
initially degrade or damage trichomes/hairs around the tissue
then rinse with distilled water
 Hydrogen peroxide (H2O2) solution – 15% and 30% at 30
minutes of soaking
 Commercial bleach solution (Zonrox) – 30% and 50% at 30
minutes of soaking

Treatments using these sterilants are grouped as follows:

T1 - 0.5%/500ml fungicide solution at 24 hours; 95% ethyl

alcohol at 1 minute soaking; 15% H 2O2 at 30 minutes and
30% Zonrox at 30 minutes

T2 - 0.5%/500ml fungicide solution at 24 hours; 95% ethyl

alcohol at 1 minute soaking; 15% H 2O2 at 30 minutes and
50% Zonrox at 30 minutes

T3 - 0.5%/500ml fungicide solution at 24 hours; 95% ethyl

alcohol at 1 minute soaking; 30% H 2O2 at 30 minutes and
30% Zonrox at 30 minutes

T4 - 0.5%/500ml fungicide solution at 24 hours; 95% ethyl

alcohol at 1 minute soaking; 30% H 2O2 at 30 minutes and
50% Zonrox at 30 minutes
 4. The explants will be initially inoculated on basic Murashige and
Skoog’s (MS) medium for establishment of sterile cultures.
5. Twenty (20) samples will be used per treatment with three (3)
replicates.

Data to be gathered:

1. Percent survival and contamination will be observed 7 days and

15 days after inoculation.

B. Induction of Somatic Embryogenesis

1. Nodal segments (sterilized using the best treatment in part A)

will be induced to shoot by inoculating the explants onto MS
liquid medium supplemented with cytokinin (BAP) at 5 ppm.
2. After 6 weeks, the sprouted shoots will be used and cut into
small pieces (3-4mm).
3. The small sections will be inoculated on basal MS medium
supplemented with different concentrations of 2,4-D (10 ppm, 15
ppm and 20 ppm) in combination with 1 ppm BAP.
4. Twenty (20) samples will be used per treatment with three (3)
replicates.
5. The cultures will be placed in a dark room to control browning.

Treatments are as follows:

T1 - Basic MS medium (without growth hormone)
T2 - 10 ppm 2,4-D + 1 ppm BAP
T3 - 15 ppm 2,4-D + 1 ppm BAP
T4 - 20 ppm 2,4-D + 1 ppm BAP

Data to be gathered:

1. Callus growth and Embryogenic frequency estimate (%) per

treatment after 4 weeks inoculation

C. Subculture and Induction of Somatic Embryogenesis

1. Proliferated friable calli will be subcultured to fresh medium

every two (2) weeks and incubated in darkness.
2. Reduced concentrations of 2,4-D will be used for induction and
maturation of somatic embryogenesis.
3. Brown, unresponsive and loose-type calli will be discarded every
transfer.
4. After three (3) months of continuous transfer, friable calli which
are potentially embryogenic will be selected and regenerated.
 Data to be gathered:

1. Frequency of somatic embryogenesis formation (%) per

treatment after 3 months.

D. Somatic Embryo Germination and Plantlet Conversion

1. Somatic embryos will be transferred to MS medium

supplemented with different concentrations of BAP for plant
regeneration at 1 ppm, 2.5 ppm, 3 ppm and 5 ppm with 0.5 ppm
NAA for effective shoot and root development.
2. Twenty (20) samples will be used per treatment with three (3)
replicates.

Treatments are as follows:

T1 - Basic MS medium (without growth hormone)
T2 - 1 ppm BAP + 0.5 ppm NAA
T3 - 2.5 ppm BAP + 0.5 ppm NAA
T4 - 3 ppm BAP + 0.5 ppm NAA
T5 - 5 ppm BAP + 0.5 ppm NAA

Data to be gathered:

1. Embryogenic efficiency/Frequency of germination (%)of somatic

embryos per treatment after 2 months

Experimental Design

Completely Randomized Design (CRD) of three (3) replications

with twenty (20) subsamples will be used in each of the sterilization,
somatic embryogenesis induction and plant regeneration stage
experiment. Mean comparison will be analyzed using Least Significant
Differences (LSD) Test at 5% level of significance. Analysis of variance
(ANOVA) will also be used (Table 1).

Table 1. ANOVA table for Completely Randomized Design

Source of variation Degrees of Sums of Mean Square F

freedom squares (SS) (MS)
Treatments (Tr) t-1 SSTr SSTr/(t-1) MSTr/MSE
Experimental Error (E) t*(r-1) SSE SSE/(t*(r-1)) MSE/MSS
Sampling Error (S) t*r*(s-1) SSS SSS/(t*r*(s-1))
Total (Tot) t*r*s-1 SSTot

E. Acclimatization and Potting-out

 1. Regenerated plantlets with well-developed roots will be
transferred and gradually acclimatized or exposed to full
sunlight.
2. All plantlets taken out from the culture room will be initially
maintained in room temperature for two weeks to serve as pre-
nursery establishment procedure (1 week with loosened cap/lid
and another week with opened cap/lid).
3. After the plantlets have adjusted with room temperature
condition, plantlets will be deflasked from the culture bottles and
will be washed thoroughly to remove the remaining agar.
4. Rooted plantlets will be transferred on seedling trays/seedling
bags with 1:1 sand + coir dust and will be enclosed with plastic
to maintain high humidity during rooting.
5. After two (2) months, plantlets will be transferred to larger pots for
further growth.

Data to be gathered:

1. Percent survival will be observed 7 days, 15 days and 30 days

after transplanting
2. Health condition

Health score:

0 - Dead
1 - Poor health (yellowing, wilting, signs and symptoms of insect
and disease)
2 - Healthy (vigorous growth, green color, with slight signs
insect damage)
3 - Very healthy (very vigorous, healthy green color, free from
insect and disease infestation)

3. Cost of producing tissue-cultured plantlets from the laboratory

upto the nursery stage.

Workplan/Activity Schedule
 SCHEDULE
ACTIVITY Year 1 Year 2 Year 3
Q1 Q2 Q3 Q4
A. Procurement, Collection and
Establishment of Source of
Planting Materials
B. Laboratory Phase
Culture Establishment
1. Collection and Sterilization
2. Shoot Induction and
multiplication
3. Induction of Somatic
Embryogenesis
4. Plantlet Regeneration and
Maintenance
C. Acclimatization and Potting-out
D. Verification
E. Monitoring and Data gathering
F. Data Analysis
G.Periodical Reports

Budget Requirement (‘000)

Year 1 Y2 Y3 Total
ITEM
Q1 Q2 Q3 Q4 Total
A. Personal
Services
(1) Laboratory
30 30 30 30 120 120 120 360
Aide I
B.Maintenance
and Other
Operating
Expenses
Travel 5 5 5
Gasoline 1 2 2
Supplies and
130 20 15 10 175 40 30 245
Materials
Other
Professional
Services
C.Capital Outlay 600 600 600
Grand Total 1,212

Expected Output
 1. Protocol developed for in vitro regeneration of kawayan kiling and bolo
through induction of somatic embryogenesis (n=2).
2. Most suitable sterilization procedure developed for kawayan kiling and bolo.
3. Culture media capable of generating maximum embryogenic efficiency and
frequency of germination (%) of somatic embryos for kawayan kiling and bolo.
4. IEC materials (Brochure/Factsheet)
5. Cost of producing tissue-cultured plantlets (produced from somatic
embryogenesis) and nursery grown plantlets of kawayan kiling and bolo.

Logical Framework
 NARRATIVE SUMMARY OBJECTIVELY MEANS OF IMPORTANT
VERIFIABLE INDICATORS VERIFICATION ASSUMPTION
GOAL: Sustained supply of bamboo Government
Mass production of bamboo raw materials statistics
species
PURPOSE:

To develop an alternative way of Protocol developed for in Manual

producing planting materials of vitro regeneration using Accomplishment
bamboo somatic embryogenesis for reports
Kawayan killing and bolo.

OUTPUTS: Established a set of

procedure for:

1. Protocol developed for in

vitro regeneration of
kawayan kiling and bolo
through induction of
somatic embryogenesis
(n=2).

2. Most suitable sterilization a) effective sterilization for Accomplisment

procedure developed for
kawayan kiling and bolo.
3. Culture media capable of
generating maximum
embryogenic efficiency
and frequency of
germination (%) of
somatic embryos for
kawayan kiling and bolo.
explants (with reports
lowest/acceptable
contamination rate) IEC material
(Brochure whenever
b) growth hormone there is a significant
combination suitable for result per every
generating maximum stage)
embryogenic efficiency and
frequency of germination
(%) of somatic embryos

4. Cost of producing tissue- c) produce low cost planting

cultured plantlets
(produced from somatic
embryogenesis) and
nursery grown plantlets of
kawayan kiling and bolo.
materials and compare cost
of production of tissue
cultured to conventionally
produced Kawayan kiling

INPUTS:

1. Bambusetum LBES Bambusetum Accomplishment

2. Laboratory facilities ERDB tissue culture reports
3. Supplies and materials laboratory
4. Nursery facilities ERDB Nursery
5. Labor ERDB personnel
6. Funds Fund allocation

Literature Cited
Anis, M. and N. Ahmad. 2016. Plant Tissue Culture: Propagation, Conservation and
Crop Improvement. pp. 511-516.

Arya, S., R. Satsangi and I.D. Arya. 2008a. Large scale plant production of edible
bamboo Dendrocalamus asper through somatic embryogenesis. Journal of
American Bamboo Society, 21(1),13-23.

Department of Environment and Natural Resources (DENR), 2016. Convergence

Program to Stimulate Bamboo Development Revealed. Retrieved from
www.denr.gov.ph on November 3, 2016.

Gonzales, L., N. Cortiguerra and A. Piñol. 1992. Reforestation Species; Kawayan

Kiling and Bolo. Research Information Series on Ecosystem (RISE). Vol. 4
No. 4. pp. 3-10.

Jain, S.M., P.K. Gupta and R.J. Newton. 2000. Somatic Embryogenesis in Woody
Plants. Volume 6. pp. 216-217.

Malini, N. and C.R. Anandakumar. 2013. Micropropagation of Bamboo (Bambusa

vulgaris) through Nodal Segment. International Journal of Forestry and Crop
Improvement Vol 4 (1) pp.36-38.

Ndiaye, A., M.S. Diallo, D. Niang and Y.K. Gassama-dia. 2006. In vitro regeneration
of adult trees of Bambusa vulgaris. African Journal of Biotechnology Vol. 5
(13) pp. 1245-1248.

Pollisco, M.T. 1991. Some Factors Affecting Callus Formation and other Response of
Kawayan tinik (Bambusa blumeana Schultes f.). (Masteral Thesis, University
of the Philippines, Los Baños, Laguna). Retrieved from
http://agris.fao.org/agris-search/search.do?recordID=PH9310396.

Rao, I.U., I.V. Rao and V. Narang. 1985. Somatic embryogenesis and regeneration
from inflorescence callus of Bambusa beechyana Munro var. beechyana.
Plant Cell Rep. 5: 409-411.

Roxas, Cristina A. 2012. Handbook on Erect Bamboo Species in the Found in the
Philippines. Ecosystems Research and Development Bureau. pp.
15,22,28,42.

Singh, S.R., R. Singh, S. Kalia, S. Dalal, A.K. Dhawan and R. Kalia. 2012.
Limitations, Progress, and Prospects of Application of Biotechnological tools
in Improvement of Bamboo – A Plant with Extraordinary Qualities. Physiol
Mol Biol Plants (January-March 2013) 19(1):21-4.

Sood, A., A. Bhattacharya, M. Sharma, R.K. Sharma, H.K. Nadha, P. Sood, R.

Mehta, D. Kaur, J. Brar and P.S. Ahuja. 2013. Somatic Embryogenesis and
Agrobacterium Mediated Genetic Transformation in Bamboos. pp. 166-178.
Virtucio, F.D., C.A. Roxas and C.B, Apolinar. 1999. Kawayan [Bamboo] : The Wonder
Grass. Canopy International Vol. 25. Ecosystems Research and
Development Bureau.

Zamora, A.B., S.S. Gruezo and O.P. Damasco. 1988. Callus Induction and Plant
Regeneration from Internode Tissues of Bamboo (Dendrocalamus latiflorus
cv. Machiku. The Philippine Agriculturist 71 (1): 76-84.

Zamora, A.B., S. Gruezo. 1990. Embryo culture of bamboo (Dendrocalamus strictus

Nees.) The Philippine Agriculturist. 73(2): 199-206.