Abstract
BIBLIOGRAPHY
GALIAN, JAKE L. APRIL 2009. In Vitro Propagation of Alstroemeria hybrida
cv. Purple. Benguet State University, La Trinidad,Benguet.
Adviser: Vilma Imelda D. Alejandro, PhD
ABSTRACT
This study was conducted at the Horticulture Research and Training Institute,
Benguet State University, La Trinidad, Benguet from October 2008 to March 2009. The
study aimed to determine the suitable explant, suitable medium and develop techniques in
propagating Alstroemeria in vitro.
Shoot tip explants grown in MS + .5 ppm NAA and MS + .5 ppm BA + .5 ppm
NAA resulted to rhizogenetic cultures. This is the key to Alstroemeria in vitro
propagation. The easier the decontamination procedure for shoot tip explants makes it a
better choice as an explant for in vitro Alstroemeria propagation. Rhizome buds are
difficult to sterilize.
A more refined system must be established. Auxin-cytokinin concentrations must
be considered for further studies. Similarly, position of explant implantation in the
growing medium is very important as observed in this study.
TABLE OF CONTENTS
Page
Bibliography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
REVIEW OF LITERATURE
Description of Alstroemeria hybrida cv. Purple . . . . . . . . . . . . . . . . . . .
3
Growth Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
Explants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
MATERIALS AND METHODS
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Explants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
9
Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
Data Gathered . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
Study 1. In Vitro Propagation of Alstroemeria hybrida cv.
Purple Using Young Leaf Explants: A Verification . . . . . . . . .
12
Study 2. In Vitro Propagation of Alstroemeria hybrida cv.
ii
Purple Using Shoot Tip Explants . . . . . . . . . . . . . . . . . . . . . . . .
13
Study 3. In Vitro Propagation of Alstroemeria hybrida cv.
Purple Using Rhizome Buds . . . . . . . . . . . . . . . . . . . . . . . . . . .
16
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
iii
INTRODUCTION
Alstroemeria, a native of tropical North America is also referred to as the “Lily of
the Incas” and “Peruvian lily”. It is becoming one of the most important cut flower crop
worldwide due to its attractive flowers and excellent keeping quality. The genus was
named from the Swedish Baron Class Alstroemeria by his close friend Caralus
Linnaeues. The plant was first described by the French botanist Lous Feuilee (Anon.,
2001). This is one of the elegant exotic flower that is introduced in the Cordillera. The
exact introduction year of this crop specifically in La Trinidad, Benguet is off the record.
There are three main varieties of Alstroemeria, the butterfly type which has a shorter
stem and peduncles, the orchid type, which has longer stems and peduncles and the
aurantica and there are a lot of cultivars, but most of today’s types are hybrid.
Alstroemeria belongs to the family Amaryllidaceae. Its genus is Alstroemeria
with about 60 species, and related to the onion, daffodil, agapanthus and nerine. The plant
has an underground rhizome where shoot emerges and grow vertically from the ground.
Alstroemeria is composed of trumpet shaped 1 ½ to 2 inches in diameter, with one or
more laterals. Many blooms are grouped atop stems 24 – 86 inches long. The stems of
these cut flowers are 2 – 3 feet long and branch into four to six short pedicels, each
holding two to four flower buds.
Alstroemeria is not endemic to the Philippines. Other types of Alstroemeria loved
by enthusiasts include the sweet Laura and Petite Plum, which vary in size and flower
color. Colors include yellow, apricot, orange, pink, red, lavender, cream, white, and bi-
colors. Perhaps the most fascinating and morphological trait of the Alstroemeria and its
relatives is the fact that the leaves are resupinate that is they twist from the base so that
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
2
what appears to be the upper surface is the lower surface. This is an expensive cut flower
but farmers have nowhere to get planting materials. Importation is very expensive.
Due to the nature of the plant that is difficult to propagate vegetative by natural
means, in vitro technique makes possible mass rapid propagation of Alstroemeria. Plant
tissue culture is a technique whereby plant parts are grown in artificial environment that
can sustain its life. Plant tissue culture has the ability to produce disease free materials.
Presently because of the increasing use of the cut flower and ornamental plant as
materials for outdoor home designs, many plants have been tried and cultivated for their
beauty and economic value. Alstroemeria is one, which interests one’s eye and has the
potential to be propagated in vitro to make available plant material since importation is
very costly.
Among the many ornamental plants, Alstroemeria is one versatile crop because it
is not only used for cut flower arrangements, but also used in landscape gardening or
even a houseplant for interior design, because of its beautiful flowers and long vase life.
This study was conducted at the Tissue Culture Laboratory, Horticulture Research
and Training Institute, Benguet State University, La Trinidad, Benguet from October
2008 to March 2009. This research work aimed: 1) to determine suitable explants for in
vitro propagation of Alstroemeria; 2) to determine suitable medium for in vitro
propagation of Alstroemeria; and 3) to develop techniques in propagating Alstroemeria in
vitro.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
REVIEW OF LITERATURE
In the development of the country’s cut flower industry, in vitro culture of flower
crops has several advantages besides its usefulness in mass propagation of plants. Tissue
culture technique may be employed in the production of disease free and quality mother
stock plants. Tissue culture of ornamental crops is an expensive process and its use may
not merit its cost (Holdgate, 1977). However, it has been proven to be economically
profitable. With further research, tissue culture is of practical value to many ornamental
crops.
Hartman and Kester (1959) reported that tissue culture can be started from a
variety of plant parts which have cells that are capable of dividing. Moreover, Torres
(1989) stated that the primary goals of tissue culture techniques in ornamental plant is the
elimination of disease and production of disease-free plant materials and the rapid
production of a large number of genetically identical plants. Introduction of a new
genotype offers several advantages for a commercial operation.
Moreover, Ammirato (1990) reported that the intent of clonal propagation is to
reproduce plants of desirable qualities uniformly and in quantity and that reproduction of
tissue culture is faster than by nursery traditional method is generally acknowledged.
Tissue culture is the development of new plants in artificial medium under aseptic
condition from very small pieces of plants, such as embryos, stems, shoot tips, root tips,
callus, single cell and pollen grains. Haberlandt (1902) has used this in research
laboratories throughout the world since it was his first attempt in 1902 to isolate growth
in plant tissue culture (Hartman, 1975).
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
4
Growth Regulators
Weaver (1972) stated that plant growth regulators play a major role in plant
growth and development and that to grow in length, tissue must have received
substances. Application of growth regulators on the growing materials accelerates root
initiation and development, and increase uniform rooting (Janick, 1972).
Auxin in the media is essential for rooting from shoot tips during the early stage
of culture. Cytokinin usually inhibits root formation but induces lateral shoot and leaf
growth.
Weaver (1972) reported that the effects of cytokinins are effective for cell
division. The relative amount of auxin and cytokinin determine whether shoots or roots
develop from callus (Thorpe, 1981). Murashige (1974) reported that a medium high in
auxin and low in cytokinin stimulated rooting and reversed balance promoted shoot
formation. They explained that to enhance axillary branching in shoot tip explants by
high concentrations of cytokinin is presumably due to the suppressive action by cytokinin
on apical dominance, a manifestation attributed to auxin. Among various cytokinins used,
it is the most commonly employed proliferation culture medium to promote axillary shoot
proliferation for different species (Nehra and Karta, 1994).
Vuylsteke (1989) mentioned that plant growth regulators and development of
explants in vitro depend on their concentrations and ratio in the medium. Often, this
determines the pattern of development in culture.
Auxin in the media is essential in the rooting of shoot tip during the early stage of
culture. Cytokinin usually inhibits root formation but induces lateral shoot and leaf
growth. A negative effect of auxin was observed in the inhibition of plant development in
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
5
callus formation. The effect of cytokinins seems to be negligible and generally produces
favorable effects only when optimally balanced with auxins (PCARRD, 1975).
Culture Media
One of the most important factors governing the growth and morphogenesis of
plant tissue cultures is the composition of the culture medium. The basic nutrient
requirements of cultured plant cells are very similar to those of whole plants.
Tissue culture techniques require surgical removal of plant tissues under aseptic
conditions; special culture media has been developed for different materials. These
involve sugars, inorganic, salts, vitamins, growth regulators and organic substances. Solid
media or semi-sold media is made with the addition of agar.
A number of media have been devised for specific tissues and organs. Murashige
and Skoog (MS) (1962) medium is widely used to induce organogenesis and regeneration
of plant tissue cultures containing the desired salt composition.
Several media formulations are commonly used for the majority of cell and tissue
culture work. These media formulations include MS (1962) medium.
Sheuk and Hilderbrandt (SH) and Gamborgs (B-5) medium are all high in
macronutrients, while other media formulations contain considerably less of the
macronutrients (Torres, 1989).
Smith (1982) stated that the choice of a particular medium depends mainly on the
species of the plant, the tissues and organ to be cultured, and concentrations of hormones
are the most important considerations in preparation of the medium for monocotyledon
and dicotyledonous tissue culture.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
6
The best induction was obtained on MS (1962) medium supplemented with 10
mM Thidiazuron (TDZ) and 0.5 mM Indole butyric acid (IBA) and the shooting medium
contained MS medium with 2.2 mM 6 –bezylamino purine (BAP) (Hsueh-Shih Lin,
1998).
Explants
The first step in any successful tissue culture program is the selection of suitable
explants source. Almost any plant tissue organ can be used as explants, but the degree of
success obtained will depend upon the culture system used, the species being cultured,
and the removal of surface contaminants from the explants (Torres, 1989).
Study showed that without understanding of the growth potential within the stock
plant is likely that culture lines generated in vitro will have quite different growth
response and rooting capacity (Marks, 1990). A small piece of plant used to begin
culture has been referred to as explant. The smaller the explant, the more effective it is in
the elimination of pathogen (Meyer, 1976).
A tissue in micropropagation is called explant. Explants range in size from 1mm
to stem pieces 0.2 inch or more (Kyte, 1983). They are usually young tissues consisting
of immature cells (Vidalie, 1995) which are composed of differentiated tissues such as
fragment of stem, roots, flowers and fruits.
Source of explants should be free from diseases and should be growing
vigorously. Meristems, shoot tips, anthers, stems, flowers, leaves, embryos, hypocotyls,
seedlings, rhizomes, bulbscales, flower buds, corms or roots can serve as explants or
sources of explants (Kyte, 1983).
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
7
Alstroemeria can be propagated in vitro by variety of techniques. One is through
the use of leaves and rhizomes (Hsueh-Shih Lin, 1998).
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
MATERIALS AND METHODS
Materials
Culture vessels, sterile petri dishes, graduated cylinder, analytical balance,
beakers, forceps, scalpels, alcohol lamp, pressure cooker, laminar flow cabinet,
laboratory gown, cap and other laboratory equipment were very important for the work.
Studies
For studies 1 and 2, there were 5 treatments, each replicated 3 times with 30
samples. Study 3 on the other hand, consisted of 4 treatments. Studies were laid out
using the Complete Randomized Experimental Design (CRD).
The different studies conducted were:
Study 1. In Vitro Propagation of Alstroemeria hybrida cv. Purple Using Young
Leaf Explants: A Verification
Study 2. In Vitro Propagation of Alstroemeria hybrida cv. Purple Using Shoot Tip
Explants
Study 3. In Vitro Propagation of Alstroemeria hybrida cv. Purple Using Rhizome
Buds
Treatments
MS 1962 basal medium enriched with different growth regulators which served as
treatments were as follows:
T1
- MS Basal medium
T2
- MS + .5 ppm Benzyl-amino purine (BA)
T3
- MS + .5 ppm Napthalene acetic acid (NAA)
T4
- MS + 1 ppm BA + .5 ppm NAA
T5
- MS + .5ppm BA + .5 ppm NAA
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
9
Explants
Young leaves, shoot tips and rhizome buds of Alstroemeria hybrida cv. Purple
were used as explants in the different studies.
Sterilization
Young leaf and shoot tip explants. Young leaves and shoot tips ere isolated from
the mother plant and thoroughly washed with detergent and running water. Final
washing was done with distilled water. This was followed by sterilization with 5% (v/v)
sodium hypochlorite for a period of 10 minutes. Sterile distilled water was used to wash
thoroughly the explants. Final sterilization was done with 1% (v/v) sodium hypochlorite
for 1 minute, after which explant was thoroughly washed with sterile distilled water.
Excess water was removed from the explant making use of sterile tissue paper.
Implantation followed.
Rhizome buds. Rhizome buds were isolated from mother plant and thoroughly
washed with detergent and running water. Rhizome buds were then soaked with 100%
sodium hypochlorite for 20 minutes followed by thorough washing with sterile distilled
water. Inside the laminar flow cabinet, sterilization was repeated using 100% sodium
hypochlorite for another 20 minutes. Again, rhizome buds were thoroughly washed with
sterile distilled water. Excess water were removed with sterile tissue paper. Implantation
followed to the respective culture medium.
Maintenance
Cultures were maintained under in a diffused light condition at ambient
temperature.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
10
Data Gathered
1. Number of leaves. Numbers of leaves were counted 60 days from implantation
(DFI).
2. Number of shoots. Numbers of shoots were counted 60 DFI.
3. Height of shoots (cm). Heights of shoots were taken 60 DFI.
4. Number of tubers. Numbers of tubers were counted 60 DFI.
5. Number of rhizogenetic cultures 60 DFI.
6. Documentation
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
RESULTS AND DISCUSSION
This section presents the results and observations from the different studies
conducted. Figures are also presented.
Figure 1 illustrates the morphological structure of an Alstroemeria hybrida cv.
Purple. This is presented to help us understand the results.
Shoot
Rhizome Bud
Root
Tuber
Figure 1 . Morphological structure of an Alstroemeria hybrida cv. Purple
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
12
Study 1. In Vitro Propagation of Alstroemeria hybrida cv.
Purple Using Young Leaf Explants: A Verification
Table 1 shows that in vitro propagation using Alstroemeria young leaf explants is
not possible. This confirms the results obtained and reported by Alejandro (2008).
On the contrary, Hsueh-Shi Lin (1998) reported that new shoots were formed
from in vitro grown Alstroemeria leaf explants containing a leaf blade and a stem node.
The best induction medium was obtained from MS (1962) medium supplemented with 10
µM thidiazuron (TDZ) and .5µM indole butyric acid (IBA).
The unavailability of TDZ during the conduct of the study prompted the use of
BA which is known to be very effective in tissue cultures for shoot formation.
Table 1. Morphogenetic response of young leaf cultures
TREATMENT
MORPHOGENETIC RESPONSE
MS
Swelling no callus formation, no shoot formation,
ultimately turned brown
MS + .5 ppm BA Swelling no callus formation, no shoot formation,
ultimately turned brown
MS + .5 ppm NAA Swelling no callus formation, no shoot formation,
ultimately turned brown
MS + 1 ppm BA + .5 ppm NAA Swelling no callus formation, no shoot formation,
ultimately turned brown
MS + .5 ppm BA + .5 ppm NAA Swelling no callus formation, no shoot formation,
ultimately turned brown
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
13
Study 2. In Vitro Propagation of Alstroemeria hybrida cv.
Purple Using Shoot Tip Explants
Study 2 revealed that shoot tip explants are good for in vitro propagation of
Alstroemeria.
Morphogenetic response of shoot tip cultures 60 DFI is summarized and
presented in Table 2.
Significant results achieved in this study are the rhizogenetic cultures. These
occurred on cultures grown in MS + .5 ppm NAA and MS + .5 ppm BA + .5 ppm NAA
(Figures 2 and 3). These could serve as the key to in vitro propagation of Alstroemeria.
It was not significantly different from the other treatments but this is a great result.
Further study must be conducted considering the position of the explant implanted. The
basal portion of the shoot tip explants should be inserted on the medium. As observed,
rhizogenetic cultures resulted when the base of shoot tip explant was implanted unto the
medium.
Moreover, BA and NAA concentrations could play significant roles as indicated
by the results. As had been reported many times, the relative amount of auxin and
cytokinin determine whether shoots or roots develop (Thorpe, 1981).
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
Table 2. Morphogenetic response of shoot tip cultures 60 DFI*
NUMBER NUMBER HEIGHT OF
NUMBER
NUMBER OF
TREATMENT
OF
OF
SHOOTS
OF
RHIZOGENETIC
REMARKS
LEAVES
SHOOTS
(cm)
TUBERS
CULTURES
MS
4.63b
1a
1.84a
0b
0a
MS+.5 ppm BA
9.40a
1a
3.60a
0b
0a
MS+.5 ppm NAA
2.70b
1a
2.53a
1.33a
0.13a
Key to
Alstroemeria
in vitro propagation
MS + 1 ppm BA + .5 ppm NAA 9.7 0a
1a
3.80a
0b
0a
MS +.5 ppm BA + .5 ppm NAA 10.03a
1a
4.03a
0.03b
0.03a
Key to
Alstroemeria
in vitro propagation
*Means with common letters are not significantly different at 0.05% level DMRT.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
15
T1 T2 T3 T4 T5
Figure 2 . Morphogenetic response of shoot tip cultures 60 DFI. T1 – MS, T2 – MS +.5
ppm BA, T3 – MS + .5 ppm NAA, T4 – MS + 1 ppm BA + .5 ppm NAA, T5 –
MS + .5 ppm BA + .5 ppm NAA
Figure 3. A close up view of the rhizogenetic response of shoot tip explant cultured in
MS+ .5 ppm NAA 60 DFI
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
16
Study 3. In Vitro Propagation of Alstroemeria hybrida
cv. Purple Using Rhizome Buds
Rhizome buds have been reported as source of explants for in vitro propagation of
Alstroemeria. This has been tried in this study. Table 3 shows the morphogenetic
response of rhizome bud cultures 60 DFI.
Rhizome buds grown in MS + 1ppm BA produced the longest shoots, most
number of leaves and most number of shoots. Significant differences were observed
among treatments. However, longer durations for observations and conduct of the study
is important. The very hard disinfestation making use of rhizome buds limited the time
span for this study. Many times, contamination as high as 100% occurred. The
researcher refused to make use of mercuric chloride as sterilant as had been used and
reported by many workers because it is hazardous to health. Moreover, the laboratory
used does not have proper sewerage system.
Table 3. Morphogenetic response of rhizome bud cultures 60 DFI*
TREATMENT
NUMBER OF
NUMBER OF
HEIGHT OF
LEAVES
SHOOTS
SHOOTS (cm)
MS
8.16ab
2.61a
3.83ab
MS+.5 ppm BA
4.66
b
2.33a
2.16b
MS + 1 ppm BA + .5 ppm
17.33a
4.00a
5.66a
NAA
MS + .5 ppm BA + .5 ppm
2.44b
3.00a
2.08b
NAA
*Means with common letter are not significantly different at 0.05% DMRT.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
17
T
1 T2 T3 T4
Figure 4. Morphogenetic response of young rhizome buds 60 DFI. T1– MS, T2 – MS +.5
ppm BA, T3 – MS + .5 ppm NAA, T4 – MS + 1 ppm BA + .5 ppm NAA, T5 –
MS + .5 ppm BA + .5 ppm NAA
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary
This study was conducted at the Tissue Culture Laboratory, Horticulture Research
and Training Institute, Benguet State University, La Trinidad, Benguet from October
2008 to March 2009. This research undertaking aimed to determine the suitable explant,
suitable medium and develop techniques in propagating Alstroemeria in vitro.
Results showed that shoot tips and rhizome buds are possible explants for in vitro
propagation of Alstroemeria.
For shoot tip explants, MS + .5 ppm NAA and MS + .5 ppm BA+ .5 ppm NAA
resulted to rhizogenetic cultures which is the key for in vitro propagation of Alstroemeria.
The very hard disinfestations of rhizome buds would make a worker choose the
use of shoot tips as explant.
Conclusions
Based on the results obtained, Alstroemeria can be propagated in vitro. Shoot tips
and rhizome buds could served as explants. The easier disinfestation procedure for shoot
tip explants makes one decide to choose as explant.
Recommendations
It is highly recommended that further studies on in vitro propagation of
Alstroemeria making use of shoot tip explants should be conducted. A refined system
should be established. As found in this study, in vitro propagation of Alstroemeria is
very possible. Optimum cytokinin and auxin concentrations must be studied and
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
19
established. Likewise, scaling up propagules in vitro is important. Hardening and
acclimatization of in vitro plantlets must also be considered.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
LITERATURE CITED
ALEJANDRO, V.D. 2008. In Vitro Propagation of Alstroemeria. A paper presented
during the BSU In-House Review. NPRCRTC. La Trinidad, Benguet.
AMMIRATO, P.V., D.A. EVANS, W.R. SHARP and Y.P.S.BAJAJ (eds). 1990.
Handbook of Plant Cell Culture Vol. 5. Mc Graw-Hill Inc., New York. Pp.82-
123.
ANONYMOUS, 2001. Flower Season. Retrieved from March 2004.
http://www.flower.feedback.com/index.php.
GABRYSZEWSKA, E. and M. HEMPEL. 1985. The influence of cytokinins and auxins
on Alstroemeria in tissue culture. Acta Hort. 167, 295-300.
HARTMAN, H.T. and D.E. KESTER. 1959. Plant Propagation: Principles and Practices.
3rd Edition. Prentice-Hall Inc., Englewood Cliffs, New Jersey. Pp. 509-525.
HARTMAN, H.T. and D.E. KESTER. 1975. Plant Propagation: Principles and Practices.
5th Edition. Prentice Hall Inc., Englewood Cliffs, New Jersey. Pp. 273-276.
HOLDGATE, D.P. 1977. Propagation of ornamentals by tissue culture. pp. 18-43 in J.
Reinert and Y.P.S Bajaj (eds). 1977. Applied and Fundamental Aspects of Plant.
Cell Tissue and Organ Culture. Springer-Verlag, Berlin.
HSUEH – SHIH LIN. 1998. Development of Two In Vitro Regeneration Systems
Through Leaf Explant and Callus Culture and the Application for Genetic
Transformation in Alstroemeria. Dissertation. Wageningen University, The
Netherlands. 2474 Summary. Pp. 1-4.
JANICK, J. 1972. Horticultural Science. 2nd Edition. W.H. Freeman and Company, New
Jersey. 509 pp.
KYTE, L. 1983. Plants from Test Tubes. An Introduction to Micropropagation. The
Timber Press, Beaverton, Oregon. ISBN 0-917 304-50-0. P. 98.
MARKS, T. R. 1990. Implication of Explants Selection Procedures in Micropropagation.
Horticultural Research Institute. Annual Report.
MEYER, M.M. Jr. 1976a. Propagation of Hosta by in vitro techniques. HortScience. 11:
309. (Abst. 137).
MEYER, M.M. Jr. 1976b. Propagation of day lilies by tissue culture. HortScience
II, 485-87.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
21
MURASHIGE, T. 1974. Plant Propagation through tissue culture. Ann. Rev. Plant
Physiol. 25, 135-166.
NEHRA, N.S. and K.K. KARTHA. 1994. Meristem and Shoot Tip Culture:
Requirements and Applications. In: Vasil IK, Thorpe T (eds). Plant Cell Tissue
Culture. Kluwer Academy. Pp. 37-70.
PCARRD. 1975. Coconut Production. Pp. 19-25.
SMITH, H. 1982. Light quantity, photoperception and plant strategy. Annual Rev. Plant
Physiol. 33: 481 – 518.
THORPE, T.A. 1981. (ed). Plant Tissue Culture. Methods and Applications in
Agriculture. Academic Press. New York, London, Toronto, Sydney. Pp. 27-41.
TORRES, K.C. (Ed.) 1989. Tissue Culture Techniques for Horticultural Crops. Van
Nostrand Reinhold. New York, 285 pp.
VIDALIE, H. 1995. In Vitro Culture and Its Application in Horticulture. Oxford and IBH
Publishing Co. Private Ltd. Pp. 16-18.
VUYLSTEKE, D.R. 1989. Shoot tip Culture for the Propagation, Conservation and
Exchange of Musa Germplasm. Practical Manuals for Handling Crop Germplasm
In Vitro. International Board for Plant Genetic Resources, Rome.
WEAVER, R.J. 1972. Plant Growth Substances in Agriculture. W.H. Freeman, and Co.,
Calf. 594 pp.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
APPENDICES
Appendix Table 1. Number of leaves produced from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
5.3
5.1
3.5
13.9
4.63
T2
11.1
8.5
8.6
28.2
9.40
T3
3.0
2.7
2.4
8.1
2.70
T4
13.3
6.7
9.2
29.2
9.70
T5
13.1
7.0
10.0
30.1
10.03
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
4
138.220
34.555
7.309*
.005
groups
Within groups
10
47.2820
4.728
Total
14
185.280
*Significant Coefficient of variation =29.79%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
23
Appendix Table 2. Number of shoots produced from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
1
1
1
3
1
T2
1
1
1
3
1
T3
1
1
1
3
1
T4
1
1
1
3
1
T5
1
1
1
3
1
No variance within groups – statistics for Table 6, cannot be computed.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
24
Appendix Table 3. Height of shoots (cm) from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
2.35
2.08
1.10
5.53
1.84
T2
4.88
3.03
2.90
10.81
3.60
T3
1.65
4.70
1.25
7.60
2.53
T4
4.91
2.85
3.65
11.41
3.80
T5
6.00
3.30
2.80
12.10
4.03
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
4
10.498
2.625
1.917ns
.298
groups
Within groups
10
18.524
1.852
Total
14
29.022
nsnot significant
Coefficient of variation=43.02%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
25
Appendix Table 4. Number of tubers produced from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
0
0
0
0
0
T2
0
0
0
0
0
T3
2.2
1.8
0
4
1.33
T4
0
0
0
0
0
T5
0
0
0.1
0.1
0.03
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
4
4.216
1.054
3.828*
0.39
groups
Within groups
10
2.753
.275
Total
14
6.969
*Significant Coefficient of variation=191.88%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
26
Appendix Table 5. Number of rhizogenetic cultures from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
0
0
0
0
0
T2
0
0
0
0
0
T3
0.1
0.3
1
0.40
0.13
T4
0
0
0
0
0
T5
0
0
0.1
0.01
0.03
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
4
0.40
0.10
1.875ns
.191
groups
Within groups
10
0.053
.005
Total
14
.093
nsnot significant
Coefficient of variation=2.12%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
27
Appendix Table 6. Number of leaves produced from rhizome bud explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
13.5
6.5
4.50
29.50
8.16
T2
2.0
10.0
2.00
14.00
4.66
T4
12.0
10.0
30.00
52.00
17.33
T5
3.0
3.0
1.33
7.33
2.44
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
3
387.101
129.034
3.111ns
.089
groups
Within groups
8
331.859
41.482
Total
11
718.960
nsnot significant
Coefficient of variation=79.03%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
28
Appendix Table 7. Number of shoots produced from rhizome bud explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
3.5
3.0
1.33
7.85
2.61
T2
2.0
3.0
2.00
7.00
2.33
T4
3.0
4.0
5.00
12.00
4.00
T5
3.5
3.5
2.00
9.00
3.00
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
3
4.787
1.596
1.189ns
.209
groups
Within groups
8
6.749
.844
Total
11
11.536
nsnot significant
Coefficient of variation=30.73%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
29
Appendix Table 8. Height (cm) of shoots produced from rhizome bud explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
6.50
2.50
2.25
11.5
3.83
T2
2.00
2.50
2.00
6.50
2.16
T4
3.00
7.50
6.50
17.00
5.66
T5
1.75
2.25
2.25
6.25
2.08
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
3
25.724
8.575
3.095ns
.089
groups
Within groups
8
22.167
2.791
Total
11
47.891
nsnot significant
Coefficient of variation=79.03%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
GALIAN, JAKE L. APRIL 2009. In Vitro Propagation of Alstroemeria hybrida
cv. Purple. Benguet State University, La Trinidad,Benguet.
Adviser: Vilma Imelda D. Alejandro, PhD
ABSTRACT
This study was conducted at the Horticulture Research and Training Institute,
Benguet State University, La Trinidad, Benguet from October 2008 to March 2009. The
study aimed to determine the suitable explant, suitable medium and develop techniques in
propagating Alstroemeria in vitro.
Shoot tip explants grown in MS + .5 ppm NAA and MS + .5 ppm BA + .5 ppm
NAA resulted to rhizogenetic cultures. This is the key to Alstroemeria in vitro
propagation. The easier the decontamination procedure for shoot tip explants makes it a
better choice as an explant for in vitro Alstroemeria propagation. Rhizome buds are
difficult to sterilize.
A more refined system must be established. Auxin-cytokinin concentrations must
be considered for further studies. Similarly, position of explant implantation in the
growing medium is very important as observed in this study.
TABLE OF CONTENTS
Page
Bibliography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
REVIEW OF LITERATURE
Description of Alstroemeria hybrida cv. Purple . . . . . . . . . . . . . . . . . . .
3
Growth Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
Explants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
MATERIALS AND METHODS
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Explants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
9
Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
Data Gathered . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
Study 1. In Vitro Propagation of Alstroemeria hybrida cv.
Purple Using Young Leaf Explants: A Verification . . . . . . . . .
12
Study 2. In Vitro Propagation of Alstroemeria hybrida cv.
ii
Purple Using Shoot Tip Explants . . . . . . . . . . . . . . . . . . . . . . . .
13
Study 3. In Vitro Propagation of Alstroemeria hybrida cv.
Purple Using Rhizome Buds . . . . . . . . . . . . . . . . . . . . . . . . . . .
16
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
18
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
iii
INTRODUCTION
Alstroemeria, a native of tropical North America is also referred to as the “Lily of
the Incas” and “Peruvian lily”. It is becoming one of the most important cut flower crop
worldwide due to its attractive flowers and excellent keeping quality. The genus was
named from the Swedish Baron Class Alstroemeria by his close friend Caralus
Linnaeues. The plant was first described by the French botanist Lous Feuilee (Anon.,
2001). This is one of the elegant exotic flower that is introduced in the Cordillera. The
exact introduction year of this crop specifically in La Trinidad, Benguet is off the record.
There are three main varieties of Alstroemeria, the butterfly type which has a shorter
stem and peduncles, the orchid type, which has longer stems and peduncles and the
aurantica and there are a lot of cultivars, but most of today’s types are hybrid.
Alstroemeria belongs to the family Amaryllidaceae. Its genus is Alstroemeria
with about 60 species, and related to the onion, daffodil, agapanthus and nerine. The plant
has an underground rhizome where shoot emerges and grow vertically from the ground.
Alstroemeria is composed of trumpet shaped 1 ½ to 2 inches in diameter, with one or
more laterals. Many blooms are grouped atop stems 24 – 86 inches long. The stems of
these cut flowers are 2 – 3 feet long and branch into four to six short pedicels, each
holding two to four flower buds.
Alstroemeria is not endemic to the Philippines. Other types of Alstroemeria loved
by enthusiasts include the sweet Laura and Petite Plum, which vary in size and flower
color. Colors include yellow, apricot, orange, pink, red, lavender, cream, white, and bi-
colors. Perhaps the most fascinating and morphological trait of the Alstroemeria and its
relatives is the fact that the leaves are resupinate that is they twist from the base so that
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
2
what appears to be the upper surface is the lower surface. This is an expensive cut flower
but farmers have nowhere to get planting materials. Importation is very expensive.
Due to the nature of the plant that is difficult to propagate vegetative by natural
means, in vitro technique makes possible mass rapid propagation of Alstroemeria. Plant
tissue culture is a technique whereby plant parts are grown in artificial environment that
can sustain its life. Plant tissue culture has the ability to produce disease free materials.
Presently because of the increasing use of the cut flower and ornamental plant as
materials for outdoor home designs, many plants have been tried and cultivated for their
beauty and economic value. Alstroemeria is one, which interests one’s eye and has the
potential to be propagated in vitro to make available plant material since importation is
very costly.
Among the many ornamental plants, Alstroemeria is one versatile crop because it
is not only used for cut flower arrangements, but also used in landscape gardening or
even a houseplant for interior design, because of its beautiful flowers and long vase life.
This study was conducted at the Tissue Culture Laboratory, Horticulture Research
and Training Institute, Benguet State University, La Trinidad, Benguet from October
2008 to March 2009. This research work aimed: 1) to determine suitable explants for in
vitro propagation of Alstroemeria; 2) to determine suitable medium for in vitro
propagation of Alstroemeria; and 3) to develop techniques in propagating Alstroemeria in
vitro.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
REVIEW OF LITERATURE
In the development of the country’s cut flower industry, in vitro culture of flower
crops has several advantages besides its usefulness in mass propagation of plants. Tissue
culture technique may be employed in the production of disease free and quality mother
stock plants. Tissue culture of ornamental crops is an expensive process and its use may
not merit its cost (Holdgate, 1977). However, it has been proven to be economically
profitable. With further research, tissue culture is of practical value to many ornamental
crops.
Hartman and Kester (1959) reported that tissue culture can be started from a
variety of plant parts which have cells that are capable of dividing. Moreover, Torres
(1989) stated that the primary goals of tissue culture techniques in ornamental plant is the
elimination of disease and production of disease-free plant materials and the rapid
production of a large number of genetically identical plants. Introduction of a new
genotype offers several advantages for a commercial operation.
Moreover, Ammirato (1990) reported that the intent of clonal propagation is to
reproduce plants of desirable qualities uniformly and in quantity and that reproduction of
tissue culture is faster than by nursery traditional method is generally acknowledged.
Tissue culture is the development of new plants in artificial medium under aseptic
condition from very small pieces of plants, such as embryos, stems, shoot tips, root tips,
callus, single cell and pollen grains. Haberlandt (1902) has used this in research
laboratories throughout the world since it was his first attempt in 1902 to isolate growth
in plant tissue culture (Hartman, 1975).
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
4
Growth Regulators
Weaver (1972) stated that plant growth regulators play a major role in plant
growth and development and that to grow in length, tissue must have received
substances. Application of growth regulators on the growing materials accelerates root
initiation and development, and increase uniform rooting (Janick, 1972).
Auxin in the media is essential for rooting from shoot tips during the early stage
of culture. Cytokinin usually inhibits root formation but induces lateral shoot and leaf
growth.
Weaver (1972) reported that the effects of cytokinins are effective for cell
division. The relative amount of auxin and cytokinin determine whether shoots or roots
develop from callus (Thorpe, 1981). Murashige (1974) reported that a medium high in
auxin and low in cytokinin stimulated rooting and reversed balance promoted shoot
formation. They explained that to enhance axillary branching in shoot tip explants by
high concentrations of cytokinin is presumably due to the suppressive action by cytokinin
on apical dominance, a manifestation attributed to auxin. Among various cytokinins used,
it is the most commonly employed proliferation culture medium to promote axillary shoot
proliferation for different species (Nehra and Karta, 1994).
Vuylsteke (1989) mentioned that plant growth regulators and development of
explants in vitro depend on their concentrations and ratio in the medium. Often, this
determines the pattern of development in culture.
Auxin in the media is essential in the rooting of shoot tip during the early stage of
culture. Cytokinin usually inhibits root formation but induces lateral shoot and leaf
growth. A negative effect of auxin was observed in the inhibition of plant development in
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
5
callus formation. The effect of cytokinins seems to be negligible and generally produces
favorable effects only when optimally balanced with auxins (PCARRD, 1975).
Culture Media
One of the most important factors governing the growth and morphogenesis of
plant tissue cultures is the composition of the culture medium. The basic nutrient
requirements of cultured plant cells are very similar to those of whole plants.
Tissue culture techniques require surgical removal of plant tissues under aseptic
conditions; special culture media has been developed for different materials. These
involve sugars, inorganic, salts, vitamins, growth regulators and organic substances. Solid
media or semi-sold media is made with the addition of agar.
A number of media have been devised for specific tissues and organs. Murashige
and Skoog (MS) (1962) medium is widely used to induce organogenesis and regeneration
of plant tissue cultures containing the desired salt composition.
Several media formulations are commonly used for the majority of cell and tissue
culture work. These media formulations include MS (1962) medium.
Sheuk and Hilderbrandt (SH) and Gamborgs (B-5) medium are all high in
macronutrients, while other media formulations contain considerably less of the
macronutrients (Torres, 1989).
Smith (1982) stated that the choice of a particular medium depends mainly on the
species of the plant, the tissues and organ to be cultured, and concentrations of hormones
are the most important considerations in preparation of the medium for monocotyledon
and dicotyledonous tissue culture.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
6
The best induction was obtained on MS (1962) medium supplemented with 10
mM Thidiazuron (TDZ) and 0.5 mM Indole butyric acid (IBA) and the shooting medium
contained MS medium with 2.2 mM 6 –bezylamino purine (BAP) (Hsueh-Shih Lin,
1998).
Explants
The first step in any successful tissue culture program is the selection of suitable
explants source. Almost any plant tissue organ can be used as explants, but the degree of
success obtained will depend upon the culture system used, the species being cultured,
and the removal of surface contaminants from the explants (Torres, 1989).
Study showed that without understanding of the growth potential within the stock
plant is likely that culture lines generated in vitro will have quite different growth
response and rooting capacity (Marks, 1990). A small piece of plant used to begin
culture has been referred to as explant. The smaller the explant, the more effective it is in
the elimination of pathogen (Meyer, 1976).
A tissue in micropropagation is called explant. Explants range in size from 1mm
to stem pieces 0.2 inch or more (Kyte, 1983). They are usually young tissues consisting
of immature cells (Vidalie, 1995) which are composed of differentiated tissues such as
fragment of stem, roots, flowers and fruits.
Source of explants should be free from diseases and should be growing
vigorously. Meristems, shoot tips, anthers, stems, flowers, leaves, embryos, hypocotyls,
seedlings, rhizomes, bulbscales, flower buds, corms or roots can serve as explants or
sources of explants (Kyte, 1983).
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
7
Alstroemeria can be propagated in vitro by variety of techniques. One is through
the use of leaves and rhizomes (Hsueh-Shih Lin, 1998).
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
MATERIALS AND METHODS
Materials
Culture vessels, sterile petri dishes, graduated cylinder, analytical balance,
beakers, forceps, scalpels, alcohol lamp, pressure cooker, laminar flow cabinet,
laboratory gown, cap and other laboratory equipment were very important for the work.
Studies
For studies 1 and 2, there were 5 treatments, each replicated 3 times with 30
samples. Study 3 on the other hand, consisted of 4 treatments. Studies were laid out
using the Complete Randomized Experimental Design (CRD).
The different studies conducted were:
Study 1. In Vitro Propagation of Alstroemeria hybrida cv. Purple Using Young
Leaf Explants: A Verification
Study 2. In Vitro Propagation of Alstroemeria hybrida cv. Purple Using Shoot Tip
Explants
Study 3. In Vitro Propagation of Alstroemeria hybrida cv. Purple Using Rhizome
Buds
Treatments
MS 1962 basal medium enriched with different growth regulators which served as
treatments were as follows:
T1
- MS Basal medium
T2
- MS + .5 ppm Benzyl-amino purine (BA)
T3
- MS + .5 ppm Napthalene acetic acid (NAA)
T4
- MS + 1 ppm BA + .5 ppm NAA
T5
- MS + .5ppm BA + .5 ppm NAA
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
9
Explants
Young leaves, shoot tips and rhizome buds of Alstroemeria hybrida cv. Purple
were used as explants in the different studies.
Sterilization
Young leaf and shoot tip explants. Young leaves and shoot tips ere isolated from
the mother plant and thoroughly washed with detergent and running water. Final
washing was done with distilled water. This was followed by sterilization with 5% (v/v)
sodium hypochlorite for a period of 10 minutes. Sterile distilled water was used to wash
thoroughly the explants. Final sterilization was done with 1% (v/v) sodium hypochlorite
for 1 minute, after which explant was thoroughly washed with sterile distilled water.
Excess water was removed from the explant making use of sterile tissue paper.
Implantation followed.
Rhizome buds. Rhizome buds were isolated from mother plant and thoroughly
washed with detergent and running water. Rhizome buds were then soaked with 100%
sodium hypochlorite for 20 minutes followed by thorough washing with sterile distilled
water. Inside the laminar flow cabinet, sterilization was repeated using 100% sodium
hypochlorite for another 20 minutes. Again, rhizome buds were thoroughly washed with
sterile distilled water. Excess water were removed with sterile tissue paper. Implantation
followed to the respective culture medium.
Maintenance
Cultures were maintained under in a diffused light condition at ambient
temperature.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
10
Data Gathered
1. Number of leaves. Numbers of leaves were counted 60 days from implantation
(DFI).
2. Number of shoots. Numbers of shoots were counted 60 DFI.
3. Height of shoots (cm). Heights of shoots were taken 60 DFI.
4. Number of tubers. Numbers of tubers were counted 60 DFI.
5. Number of rhizogenetic cultures 60 DFI.
6. Documentation
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
RESULTS AND DISCUSSION
This section presents the results and observations from the different studies
conducted. Figures are also presented.
Figure 1 illustrates the morphological structure of an Alstroemeria hybrida cv.
Purple. This is presented to help us understand the results.
Shoot
Rhizome Bud
Root
Tuber
Figure 1 . Morphological structure of an Alstroemeria hybrida cv. Purple
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
12
Study 1. In Vitro Propagation of Alstroemeria hybrida cv.
Purple Using Young Leaf Explants: A Verification
Table 1 shows that in vitro propagation using Alstroemeria young leaf explants is
not possible. This confirms the results obtained and reported by Alejandro (2008).
On the contrary, Hsueh-Shi Lin (1998) reported that new shoots were formed
from in vitro grown Alstroemeria leaf explants containing a leaf blade and a stem node.
The best induction medium was obtained from MS (1962) medium supplemented with 10
µM thidiazuron (TDZ) and .5µM indole butyric acid (IBA).
The unavailability of TDZ during the conduct of the study prompted the use of
BA which is known to be very effective in tissue cultures for shoot formation.
Table 1. Morphogenetic response of young leaf cultures
TREATMENT
MORPHOGENETIC RESPONSE
MS
Swelling no callus formation, no shoot formation,
ultimately turned brown
MS + .5 ppm BA Swelling no callus formation, no shoot formation,
ultimately turned brown
MS + .5 ppm NAA Swelling no callus formation, no shoot formation,
ultimately turned brown
MS + 1 ppm BA + .5 ppm NAA Swelling no callus formation, no shoot formation,
ultimately turned brown
MS + .5 ppm BA + .5 ppm NAA Swelling no callus formation, no shoot formation,
ultimately turned brown
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
13
Study 2. In Vitro Propagation of Alstroemeria hybrida cv.
Purple Using Shoot Tip Explants
Study 2 revealed that shoot tip explants are good for in vitro propagation of
Alstroemeria.
Morphogenetic response of shoot tip cultures 60 DFI is summarized and
presented in Table 2.
Significant results achieved in this study are the rhizogenetic cultures. These
occurred on cultures grown in MS + .5 ppm NAA and MS + .5 ppm BA + .5 ppm NAA
(Figures 2 and 3). These could serve as the key to in vitro propagation of Alstroemeria.
It was not significantly different from the other treatments but this is a great result.
Further study must be conducted considering the position of the explant implanted. The
basal portion of the shoot tip explants should be inserted on the medium. As observed,
rhizogenetic cultures resulted when the base of shoot tip explant was implanted unto the
medium.
Moreover, BA and NAA concentrations could play significant roles as indicated
by the results. As had been reported many times, the relative amount of auxin and
cytokinin determine whether shoots or roots develop (Thorpe, 1981).
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
Table 2. Morphogenetic response of shoot tip cultures 60 DFI*
NUMBER NUMBER HEIGHT OF
NUMBER
NUMBER OF
TREATMENT
OF
OF
SHOOTS
OF
RHIZOGENETIC
REMARKS
LEAVES
SHOOTS
(cm)
TUBERS
CULTURES
MS
4.63b
1a
1.84a
0b
0a
MS+.5 ppm BA
9.40a
1a
3.60a
0b
0a
MS+.5 ppm NAA
2.70b
1a
2.53a
1.33a
0.13a
Key to
Alstroemeria
in vitro propagation
MS + 1 ppm BA + .5 ppm NAA 9.7 0a
1a
3.80a
0b
0a
MS +.5 ppm BA + .5 ppm NAA 10.03a
1a
4.03a
0.03b
0.03a
Key to
Alstroemeria
in vitro propagation
*Means with common letters are not significantly different at 0.05% level DMRT.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
15
T1 T2 T3 T4 T5
Figure 2 . Morphogenetic response of shoot tip cultures 60 DFI. T1 – MS, T2 – MS +.5
ppm BA, T3 – MS + .5 ppm NAA, T4 – MS + 1 ppm BA + .5 ppm NAA, T5 –
MS + .5 ppm BA + .5 ppm NAA
Figure 3. A close up view of the rhizogenetic response of shoot tip explant cultured in
MS+ .5 ppm NAA 60 DFI
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
16
Study 3. In Vitro Propagation of Alstroemeria hybrida
cv. Purple Using Rhizome Buds
Rhizome buds have been reported as source of explants for in vitro propagation of
Alstroemeria. This has been tried in this study. Table 3 shows the morphogenetic
response of rhizome bud cultures 60 DFI.
Rhizome buds grown in MS + 1ppm BA produced the longest shoots, most
number of leaves and most number of shoots. Significant differences were observed
among treatments. However, longer durations for observations and conduct of the study
is important. The very hard disinfestation making use of rhizome buds limited the time
span for this study. Many times, contamination as high as 100% occurred. The
researcher refused to make use of mercuric chloride as sterilant as had been used and
reported by many workers because it is hazardous to health. Moreover, the laboratory
used does not have proper sewerage system.
Table 3. Morphogenetic response of rhizome bud cultures 60 DFI*
TREATMENT
NUMBER OF
NUMBER OF
HEIGHT OF
LEAVES
SHOOTS
SHOOTS (cm)
MS
8.16ab
2.61a
3.83ab
MS+.5 ppm BA
4.66
b
2.33a
2.16b
MS + 1 ppm BA + .5 ppm
17.33a
4.00a
5.66a
NAA
MS + .5 ppm BA + .5 ppm
2.44b
3.00a
2.08b
NAA
*Means with common letter are not significantly different at 0.05% DMRT.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
17
T
1 T2 T3 T4
Figure 4. Morphogenetic response of young rhizome buds 60 DFI. T1– MS, T2 – MS +.5
ppm BA, T3 – MS + .5 ppm NAA, T4 – MS + 1 ppm BA + .5 ppm NAA, T5 –
MS + .5 ppm BA + .5 ppm NAA
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
Summary
This study was conducted at the Tissue Culture Laboratory, Horticulture Research
and Training Institute, Benguet State University, La Trinidad, Benguet from October
2008 to March 2009. This research undertaking aimed to determine the suitable explant,
suitable medium and develop techniques in propagating Alstroemeria in vitro.
Results showed that shoot tips and rhizome buds are possible explants for in vitro
propagation of Alstroemeria.
For shoot tip explants, MS + .5 ppm NAA and MS + .5 ppm BA+ .5 ppm NAA
resulted to rhizogenetic cultures which is the key for in vitro propagation of Alstroemeria.
The very hard disinfestations of rhizome buds would make a worker choose the
use of shoot tips as explant.
Conclusions
Based on the results obtained, Alstroemeria can be propagated in vitro. Shoot tips
and rhizome buds could served as explants. The easier disinfestation procedure for shoot
tip explants makes one decide to choose as explant.
Recommendations
It is highly recommended that further studies on in vitro propagation of
Alstroemeria making use of shoot tip explants should be conducted. A refined system
should be established. As found in this study, in vitro propagation of Alstroemeria is
very possible. Optimum cytokinin and auxin concentrations must be studied and
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
19
established. Likewise, scaling up propagules in vitro is important. Hardening and
acclimatization of in vitro plantlets must also be considered.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
LITERATURE CITED
ALEJANDRO, V.D. 2008. In Vitro Propagation of Alstroemeria. A paper presented
during the BSU In-House Review. NPRCRTC. La Trinidad, Benguet.
AMMIRATO, P.V., D.A. EVANS, W.R. SHARP and Y.P.S.BAJAJ (eds). 1990.
Handbook of Plant Cell Culture Vol. 5. Mc Graw-Hill Inc., New York. Pp.82-
123.
ANONYMOUS, 2001. Flower Season. Retrieved from March 2004.
http://www.flower.feedback.com/index.php.
GABRYSZEWSKA, E. and M. HEMPEL. 1985. The influence of cytokinins and auxins
on Alstroemeria in tissue culture. Acta Hort. 167, 295-300.
HARTMAN, H.T. and D.E. KESTER. 1959. Plant Propagation: Principles and Practices.
3rd Edition. Prentice-Hall Inc., Englewood Cliffs, New Jersey. Pp. 509-525.
HARTMAN, H.T. and D.E. KESTER. 1975. Plant Propagation: Principles and Practices.
5th Edition. Prentice Hall Inc., Englewood Cliffs, New Jersey. Pp. 273-276.
HOLDGATE, D.P. 1977. Propagation of ornamentals by tissue culture. pp. 18-43 in J.
Reinert and Y.P.S Bajaj (eds). 1977. Applied and Fundamental Aspects of Plant.
Cell Tissue and Organ Culture. Springer-Verlag, Berlin.
HSUEH – SHIH LIN. 1998. Development of Two In Vitro Regeneration Systems
Through Leaf Explant and Callus Culture and the Application for Genetic
Transformation in Alstroemeria. Dissertation. Wageningen University, The
Netherlands. 2474 Summary. Pp. 1-4.
JANICK, J. 1972. Horticultural Science. 2nd Edition. W.H. Freeman and Company, New
Jersey. 509 pp.
KYTE, L. 1983. Plants from Test Tubes. An Introduction to Micropropagation. The
Timber Press, Beaverton, Oregon. ISBN 0-917 304-50-0. P. 98.
MARKS, T. R. 1990. Implication of Explants Selection Procedures in Micropropagation.
Horticultural Research Institute. Annual Report.
MEYER, M.M. Jr. 1976a. Propagation of Hosta by in vitro techniques. HortScience. 11:
309. (Abst. 137).
MEYER, M.M. Jr. 1976b. Propagation of day lilies by tissue culture. HortScience
II, 485-87.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
21
MURASHIGE, T. 1974. Plant Propagation through tissue culture. Ann. Rev. Plant
Physiol. 25, 135-166.
NEHRA, N.S. and K.K. KARTHA. 1994. Meristem and Shoot Tip Culture:
Requirements and Applications. In: Vasil IK, Thorpe T (eds). Plant Cell Tissue
Culture. Kluwer Academy. Pp. 37-70.
PCARRD. 1975. Coconut Production. Pp. 19-25.
SMITH, H. 1982. Light quantity, photoperception and plant strategy. Annual Rev. Plant
Physiol. 33: 481 – 518.
THORPE, T.A. 1981. (ed). Plant Tissue Culture. Methods and Applications in
Agriculture. Academic Press. New York, London, Toronto, Sydney. Pp. 27-41.
TORRES, K.C. (Ed.) 1989. Tissue Culture Techniques for Horticultural Crops. Van
Nostrand Reinhold. New York, 285 pp.
VIDALIE, H. 1995. In Vitro Culture and Its Application in Horticulture. Oxford and IBH
Publishing Co. Private Ltd. Pp. 16-18.
VUYLSTEKE, D.R. 1989. Shoot tip Culture for the Propagation, Conservation and
Exchange of Musa Germplasm. Practical Manuals for Handling Crop Germplasm
In Vitro. International Board for Plant Genetic Resources, Rome.
WEAVER, R.J. 1972. Plant Growth Substances in Agriculture. W.H. Freeman, and Co.,
Calf. 594 pp.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
APPENDICES
Appendix Table 1. Number of leaves produced from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
5.3
5.1
3.5
13.9
4.63
T2
11.1
8.5
8.6
28.2
9.40
T3
3.0
2.7
2.4
8.1
2.70
T4
13.3
6.7
9.2
29.2
9.70
T5
13.1
7.0
10.0
30.1
10.03
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
4
138.220
34.555
7.309*
.005
groups
Within groups
10
47.2820
4.728
Total
14
185.280
*Significant Coefficient of variation =29.79%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
23
Appendix Table 2. Number of shoots produced from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
1
1
1
3
1
T2
1
1
1
3
1
T3
1
1
1
3
1
T4
1
1
1
3
1
T5
1
1
1
3
1
No variance within groups – statistics for Table 6, cannot be computed.
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
24
Appendix Table 3. Height of shoots (cm) from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
2.35
2.08
1.10
5.53
1.84
T2
4.88
3.03
2.90
10.81
3.60
T3
1.65
4.70
1.25
7.60
2.53
T4
4.91
2.85
3.65
11.41
3.80
T5
6.00
3.30
2.80
12.10
4.03
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
4
10.498
2.625
1.917ns
.298
groups
Within groups
10
18.524
1.852
Total
14
29.022
nsnot significant
Coefficient of variation=43.02%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
25
Appendix Table 4. Number of tubers produced from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
0
0
0
0
0
T2
0
0
0
0
0
T3
2.2
1.8
0
4
1.33
T4
0
0
0
0
0
T5
0
0
0.1
0.1
0.03
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
4
4.216
1.054
3.828*
0.39
groups
Within groups
10
2.753
.275
Total
14
6.969
*Significant Coefficient of variation=191.88%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
26
Appendix Table 5. Number of rhizogenetic cultures from shoot tip explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
0
0
0
0
0
T2
0
0
0
0
0
T3
0.1
0.3
1
0.40
0.13
T4
0
0
0
0
0
T5
0
0
0.1
0.01
0.03
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
4
0.40
0.10
1.875ns
.191
groups
Within groups
10
0.053
.005
Total
14
.093
nsnot significant
Coefficient of variation=2.12%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
27
Appendix Table 6. Number of leaves produced from rhizome bud explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
13.5
6.5
4.50
29.50
8.16
T2
2.0
10.0
2.00
14.00
4.66
T4
12.0
10.0
30.00
52.00
17.33
T5
3.0
3.0
1.33
7.33
2.44
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
3
387.101
129.034
3.111ns
.089
groups
Within groups
8
331.859
41.482
Total
11
718.960
nsnot significant
Coefficient of variation=79.03%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
28
Appendix Table 7. Number of shoots produced from rhizome bud explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
3.5
3.0
1.33
7.85
2.61
T2
2.0
3.0
2.00
7.00
2.33
T4
3.0
4.0
5.00
12.00
4.00
T5
3.5
3.5
2.00
9.00
3.00
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
3
4.787
1.596
1.189ns
.209
groups
Within groups
8
6.749
.844
Total
11
11.536
nsnot significant
Coefficient of variation=30.73%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
29
Appendix Table 8. Height (cm) of shoots produced from rhizome bud explants 60 DFI
REPLICATION
TREATMENT
TOTAL
MEAN
I
II
II
T1
6.50
2.50
2.25
11.5
3.83
T2
2.00
2.50
2.00
6.50
2.16
T4
3.00
7.50
6.50
17.00
5.66
T5
1.75
2.25
2.25
6.25
2.08
ANALYSIS OF VARIANCE
SOURCES
DEGREES SUM OF
MEAN COMPUTED PROBABILITY
OF
OF
SQUARES SQUARE
F
VARIANCE FREEDOM
Between
3
25.724
8.575
3.095ns
.089
groups
Within groups
8
22.167
2.791
Total
11
47.891
nsnot significant
Coefficient of variation=79.03%
In Vitro Propagation of Alstroemeria hybrida cv. Purple. / Jake L. Galian. 2009
Document Outline
- In Vitro Propagation of Alstroemeria hybridacv. Purple
- BIBLIOGRAPHY
- ABSTRACT
- TABLE OF CONTENTS
- INTRODUCTION
- REVIEW OF LITERATURE
- Growth Regulators
- Culture Media
- Explants
- MATERIALS AND METHODS
- Materials
- Studies
- Treatments
- Explants
- Sterilization
- Maintenance
- Data Gathered
- RESULTS AND DISCUSSION
- Study 1. In Vitro Propagation of Alstroemeria hybrida cv.Purple Using Young Leaf Explants: A Verification
- Study 2. In Vitro Propagation of Alstroemeria hybrida cv.Purple Using Shoot Tip Explants
- Study 3. In Vitro Propagation of Alstroemeria hybridacv. Purple Using Rhizome Buds
- SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
- LITERATURE CITED
- APPENDICES