Propagation of Hevea brasiliensis Through In Vitro Techniques

Rationale

Globalization requires enhanced productivity in rubber trees by providing both high-yielding

clonal material and material adapted to non-traditional rubber growing regions. Increased
yields and productivity in rubber plantations can also be achieved through the release of new
latex and latex timber clones, the adaptation of latex harvesting technologies such as improved
tapping and ethephon stimulation, and the substitution of seedlings for clonal plantings.
Rubber trees are traditionally propagated by grafting buds from selected clones onto seedlings
from seed nurseries. It takes at least a year for the plants to be ready to be transplanted into the
field. Despite having a much higher and more consistent productivity than seedling-established
plantations, grafted clones are subject to graft-induced drawbacks such as imbalanced aerial
development, maturation-induced negative effects of the material produced by traditional
budwood gardens, and various types of scion X rootstock incompatibilities, which can be one
cause of within-clone variability and the bark necrosis syndrome (Masson and Monteuuis,
2017). The rootstock's inherent heterogeneity is the primary source of intraclonal variability in
growth vigor and yield.

To avoid rootstock-scion incompatibility, as well as to reduce plant production costs and time,
new propagation strategies should be considered. For large-scale multiplication of selected
clones, micropropagation, or in vitro propagation of entire plants, is a promising technology.
When compared to traditional vegetative propagation methods, it offers the advantages of
requiring less area and costing less money. Micro cutting propagation technique produces true-
to-type clones thereby minimizing intra-clonal variation caused by stock-scion interaction,
which is crucial for commercial rubber clone production.
Thus, this study will examine the feasibility of propagating hevea clones by somatic procedures
to generate true-to-type, disease-free, and vigorous rubber trees. The results of this research
may pave the way for the rapid generation of rubber clones with enhanced agronomic
characteristics such as increased rubber biosynthesis and timber volume, resistance to diseases,
and resilience to a variety of abiotic challenges.
Objectives
General objective:
This study aims to propagate true-to-type rubber trees through somatic propagation in the form
of shoot-tip cultures and nodal cultures.

Specific objectives:
1. Develop and optimize a protocol to successfully propagate Hevea in vitro using shoot-
tips and axillary buds
2. To grow in vitro propagated H.brasiliensis in the field until juvenile stage
3. Compare the efficiency between propagating rubber using micropropagation vis a vis
traditional technique

Expected output per objective:

1. PRRI protocol on in vitro propagation of Hevea
2. Data on the field performance of in vitro propagated H. brasiliensis
3. Comparative efficiency analysis between in vitro propagated H.brasiliensis versus
traditionally propagated rubber
Review of related literature
Current problem
Rubber seeds are not only very recalcitrant but also of low quality, low vigour potency and low
germination frequency. Low quality seed and poor germination rates affect the availability of
planting stocks. Traditionally, rubber trees are grown from budding propagation techniques
(Corpus, 2013). However, this technique has also its fair share of disadvantages. Brown budding
is generally carried out by grafting brown-colored buds taken from the budwood of about 1
year’s growth on to stock plants of 10 month’s age (Priyadarshan, 2011). This is a long period to
raise seedlings and budsticks and generally requires a large area. Moreover, the rubber yield
per ha, produced by trees grafted on heterogeneous illegitimate seedling rootstocks, has
reached its maximum (Mignon & Werbrouck, 2018).
Root heterogeneity and stock-scion interactions
The grafting technique, which allows the reproduction of elite genotypes at the bud-grafted
component level and specifies the use of clones as the nearly exclusive varietal type in rubber
cropping, is responsible for a large part of rubber breeding efficiency. Regardless, a bud-grafted
population has a high level of homogeneity and should have little intraclonal yield fluctuation,
excluding issues like soil heterogeneity, bud juvenility, and varied seedling rootstocks
(Priyadarshan, 2011).
In vitro propagation
In vitro culture research has led to three types of micropropagation techniques and genetic
modification in rubber. These are microcutting, short-term somatic embryogenesis and long-
term maintained somatic embryogenesis. Plant clonal propagation in closed vessels under
aseptic conditions is known as micropropagation. The plants are grown in vitro, which means
'in glass,' inside the containers on culture media containing nutrients and growth regulators
(Roberts & Schum, 2003).
Despite the fact that in vitro culture research began decades ago, many fields are still in their
infancy due to commercial application issues. The use of rejuvenated clonal plant material could
potentially provide important agricultural attributes such as higher growth, latex yield, and
resistance to wind and dryness, according to three considerations: cloning the root system
would generate new and more homogeneous rootstocks or monogenetic clones, selection of
clonal roots would improve the exploitation of existing genetic variability, and use of
rejuvenated clonal plant material could potentially provide important agricultural attributes
such as higher growth, latex yield, and resistance to wind (Priyadarshan, 2011).

As with mature clonal explants, nodal and shoot tip explants grown from seedlings can be used
for micropropagation in vitro. The failure to generate an appropriate tap root system, which is
required for tree stability, and the poor reactivity to culture conditions are the two main issues
with using clonal material from mature Hevea trees. In vitro micrografting has solved the latter
difficulty to a large extent. Induction of somatic embryogenesis in Hevea utilizing various
explants, medium compositions, and circumstances has recently piqued attention, particularly
for use in genetic transformation investigations. Using limited genotypes of Hevea, a few
researchers in different countries have documented successful somatic embryo development
and plant regeneration. There is yet to be a large-scale commercial application of tissue culture
techniques for mass replication of clonal Hevea, either through microcuttings or through
somatic embryogenesis. However, there has been enough development in research to suggest
that Hevea tissue culture can and should be improved further (Nayanakantha & Seneviratne,
2007).
Advantages of in vitro culture
Plant tissue in modest quantities is adequate for the generation of millions of clones per year via
micropropagation. Producing an equal amount of plants using traditional methods would take a
long period. Micropropagation is an excellent alternative for plant species that are resistant to
traditional bulk propagation methods. Any variation can be mass-produced in vast quantities,
and the time it takes to develop new types is cut in half. Micropropagation also has the benefit of
requiring less area. This aids in the conservation of endangered species and the preservation of
germplasm. As a result of employing meristem tip culture, disease-free cultivars can be obtained
(Bhatia percent Sharma, 2015).
Related studies
Shoot-tip cultures, nodal cultures, somatic embryogenesis, and genetic manipulation have all
been successful in Hevea in vitro. Microcutting involves in vitro culture of apical and axillary
buds cotyledonary nodes in Hevea brasiliensis to generate plantlets for propagation (Montoro,
et al., 2007).

Since the 1980s, France's Centre de Coopération Internationale en Recherche Agronomique

pour le Développement (CIRAD) has been working on rubber tree in vitro culture and has
developed various vegetative micropropagation procedures, including somatic embryogenesis
and microcutting of selected mature clones. Self-rooted and budded rejuvenated clones are two
novel varietal types that have been developed. Carron and his colleagues conducted field studies
on the micropropagation of several rubber clones in 2009. Six clones, IRCA 18, RRIM 600, PB
260, PB 254, PR 107, and BPM 24, were satisfactorily appraised as self-rooted rejuvenated
clones from M or PSE. For each field testing, clonal compliance was verified. In most situations,
the growth of these vitro plants was much faster than that of the mature budded control, and in
no case was it slower. Their dry rubber production is significantly higher in all three tapped
testing.
Dickson and colleagues investigated the in vitro culture of H. brasiliensis embryos using various
doses of benzylaminopurine (BAP) and various culture mediums in 2011. This experiment was
successful in identifying a good growth substrate for in-vitro production of rubber plantlets
with well-developed taproot. H. brasiliensis thrives in culture with 0.075 mg/l BAP and 0.01
mg/l NAA hormone supplementation, according to their findings.

Moradpour and Abdullah (2016) also looked into how to grow rubber in vitro from field
explants and how silver nanoparticles can help reduce contamination and browning. Explants
derived from the three leaf developmental phases of preculture treated plants had a moderate
to high proportion of explant survival, according to the researchers' findings. Furthermore, the
percentage of explants that survived across all leaf developmental stages was negatively
correlated with the quantity of NaOCl. They concluded that understanding the species leaf
developmental stages, the interaction of the explant source with the environment, and the
effective application of non-toxic silver nano particles in reducing microbial contamination and
browning of Hevea explants are all necessary for the successful establishment of in vitro culture
of H. brasiliensis.
Methodology

Collection of Planting Material

Explants will be obtained from grafted rubber clones aging between 1-3 years old grown at the
PRRI nursery. Clones to be used in the experiment will be USM 1, PB 260, and PB 330.

Induction of shoots from Hevea brasiliensis shoot-tip explants

Harvesting of explants will be done following the method of Wiredu et al, 2018. Shoot-tips of
seedlings will be harvested as explants using a scalpel blade and then put into a clean tight-lid
horny jar. The explants will be washed under running tap water for 10 minutes and then
washed with liquid detergent for 20 minutes. Thereafter, they will be sterilized by immersing in
0.1% mercuric chloride (HgCl2) for 5 minutes followed by rinsing with three changes of sterile
distilled water and cultured on a full strength Murashige and Skoog (16) basal medium
amended with 30.0g/L sucrose, 100.0mg/L myo-inositol, 2.0g/L activated charcoal, 1.0mg/L
silver nitrate , 2.0mg/L and control, 2.5, 5.0, 7.5 or 10.0mg/L kinetin. The pH of the medium will
be adjusted to 5.8±1 using 1.0M NaOH and 1.0M HCL before the addition of 3.0g/L phytagel and
then autoclave at 121˚C for 15minutes at 15psi. Cultured explants will be incubated in the
growth room at a temperature of 25±2˚C under a 16-hour photoperiod with light supplied by
fluorescent lamps/tubes at an intensity of 3000 lux.

Monitoring
The number of cuttings that developed plantlets will be counted. The number of leaves, shoots,
roots and height of developing shoots produced per explant will be monitored on a weekly basis
for a duration of 18 weeks.
Induction of shoots from Hevea brasiliensis with nodal explants
Young shoots with axillary buds of rubber seedlings will harvested as explants using a scalpel
blade and then put into a clean tight-lib horny jar. Nodal cuttings will be prepared from the
seedlings of two nodes. The nodal explants will be sterilized and cultured as previously
described. Cultures will be kept under growth room conditions. Nodal cuttings will be
considered as sprouted when the buds will rupture with at least one leaf.
Monitoring
The number of cuttings that developed plantlets will be counted. The number of leaves, shoots,
roots and height of developing shoots produced per explant will be monitored on a weekly basis
for a duration of 18 weeks.
Nursery Establishment of In Vitro Grown Hevea
Successfully propagated explants will be transferred in polybags (17.78 cm by 35.56 cm, with
thickness of 0.003 mm) and will be continually monitored in the nursery. Plants will be supplied
with fertilizer following the recommendation from Philippine National Standards-Good
Agricultural Practices for rubber. Complete fertilizer (14-14-14) of 40 g per plant should be
applied 3 days prior to planting and every month thereafter to 5 months.
Parameters
Growth parameters like plant height, girth size, and number of leaves will be taken bimonthly.
Plants will be observed for possible disease infection and pest infestations.
References
Antwi-Wiredu, Anthony & Amiteye, Samuel & Diawuoh, Rhoda & Asumeng, Alex & Klu, George.
2018. In Vitro Propagation of Rubber Tree (Hevea Brasiliensis) Using Shoot-Tip and
Nodal Cutting Explants. International Journal of Advances in Scientific Research and
Engineering. 4. 38-50. 10.31695/IJASRE.2018.32743.
Corpuz, O. S. (2013). Stem cut: An alternative propagation technology for rubber (Hevea
brasiliensis) tree species. International Journal of Biodiversity and Conservation, 5:2:78-
87.
Masson, Aurélien & Monteuuis, Olivier. (2017). Rubber tree clonal plantations: Grafted vs self-
rooted plant material. Bois et Forets des Tropiques. 332. 57-68.
10.19182/bft2017.332.a31333.
Masson, A., & Monteuuis, O. 2017. Mass production of self-rooted Hevea brasiliensis industrial
clones by tissue culture and nursery methods.
Montoro, P., Carron,M., Lardet, L., Clément-Demange, A., & Leclercq, J. 2007. Biotechnologies in
rubber tree (Hevea brasiliensis). Asian Pacific Conference on Tissue Culture and
Agribiotechnology.
Mignon, E., & Werbrouck, S. (2018). Somatic Embryogenesis as Key Technology for Shaping the
Rubber Tree of the Future. Frontiers in Plant Science, 9. doi:10.3389/fpls.2018.01804
Roberts, A., & Schum, A. 2003. Cell Tissue and Organ Culture. Encyclopedia of Rose Science.
Bhatia, S., & Sharma, K. 2015. Micropropagation. Modern Applications of Plant Biotechnology in
Pharmaceutical Sciences.
Dickson, I., Okere, A., Elizabeth, J., Mary, O., Olatunde, F., & Abiodun, S. 2011. In-vitro culture of
Hevea brasiliensis (rubber tree) embryo. Journal of Plant Breeding and Crop Science Vol.
3(9), pp. 185-189.
Moradpour, M., & Abdullah, S. 2016. Establishment of in vitro Culture of Rubber (Hevea
brasiliensis) from Field-derived Explants: Effective Role of Silver Nanoparticles in
Reducing Contamination and Browning. Journal of Nanomedicine & Nanotechnology.
Priyadarshan, P.M. 2011. Biology of Hevea Rubber. Rubber Research Institute of India. India.
Carron, M. P., Lardet, L., Leconte, A., Dea, B. G., Keli, J., Granet, F., Montoro, P. (2009). Field Trials
Network Emphasizes The Improvement Of Growth And Yield Through
Micropropagation In Rubber Tree (Hevea brasiliensis). Acta Horticulturae, (812), 485–
492. doi:10.17660/actahortic.2009.812.
Nayanakantha, N., & Seneviratne, P. 2007. Tissue culture of rubber: past, present, and future
prospects. Rubber Research Institute of Sri Lanka. Cey. J. Sci. (Bio. Sci.) 36 (2): 116-
125.
Annual Deliverables
Workplan

Propagation of Hevea brasiliensis Through In Vitro Techniques

Target Workplan Schedule

PAP/S No. of Year
Activities Year Year Year 1/Q Year Year Year Year Year Year Year Year
1/Q1 1/Q2 1/Q3 4 2/Q1 2/Q2 2/Q3 2/Q4 3/Q1 3/Q2 3/Q3 3/Q4
1. Research and Development
a. Procurement of Materials 1
b. Project Coordination with USM 1
c. Hiring of Project Assistant 1
d. Collection and Isolation of Plant
Tissue Specimens 4
e. Induction of shoots from Hevea
brasiliensis shoot-tip explants 2
f. Induction of shoots from Hevea
brasiliensis with nodal explants 1
g. Monitoring 1
h. Nursery Establishment of In Vitro
Grown Hevea 1
i. Finalization of Terminal Reports 1
Budgetary Requirements

Source of Fund: Implementing Agency: PRRI

Year 1
Item Total
Q1 Q2 Q3 Q4 Total
I. Personal Services (PS)
A. Honoraria
B. Salaries and Wages 68,394.00 68,394.00 68,394.00 68,394.00 273,576.00 273,576.00
Sub-Total for PS 68,394.00 68,394.00 68,394.00 68,394.00 273,576.00 273,576.00
II. Maintenance and Other Operating
Expenses (MOOE)
452,168.
A. Travelling Expenses 113,042.00 113,042.00 113,042.00 113,042.00
452,168.00 00
80,000.
B. Communication Expenses 20,000.00 20,000.00 20,000.00 20,000.00
80,000.00 00
359,150.
C. Supplies and Materials 293,150.00 22,000.00 22,000.00 22,000.00
359,150.00 00
570,000.
D. Professional Services 210,000.00 90,000.00
90,000.00 180,000.00 570,000.00 00
360,480.
E. Labor and Wages 90,120.00 90,120.00
90,120.00 90,120.00 360,480.00 00
50,000.
F. Repairs and Maintenance
00
G. Other Maintenance and Operating 145,000.
30,000.00 30,000.00 30,000.00 55,000.00
Expenses 145,000.00 00
756,312.00 365,162.00 415,162.00 480,162.00 2,016,798.00 2,016,798.0
Sub-Total for MOOE
0
III. Machinery and Equipment Outlay
190,000.00 190,000.00 190,000.00
(EO)
Sub-Total for EO 190,000.00 0.00 0.00 0.00 190,000.00 190,000.00
Administrative Cost
TOTAL 1,014,706.00 433,556.00 483,556.00 548,556.00 2,480,374.00 2,480,374.00

Prepared by: Noted by: Noted by:

_______________________________________
GLENN CARL V. ANDALAHAO,
ELLINE T. MACAY, PRRI PRRI DR. GIRLIE R. SALUDO
Project Leader, PRRI Accountant, PRRI Director, PRRI