Oxalis triangularis is an attractive ornamental



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AbstractOxalis triangularis is an attractive ornamental 

plant propagated by using bulbs and has no natural viable 

seeds.  It is an ideal decorative plant for growing in pots and 

other containers. The plant is not widely found in Malaysia and 

mostly obtained from Thailand. Hence, it is necessary to 

propagate this plant through tissue culture system for large 

scale production. In the present study, production of synthetic 

seeds was attempted from this species and the synthetic seeds 

managed to survive after 7 and 30 days after storage at 4°C. 

The rate of synthetic seeds conversion to complete plants after 

7 days was 96.67% with 4.57 mean shoot numbers, while after 

30 days of storage, the conversion rate slightly decreased to 

90% with only 3.97 shoots formation per bead.  The present 

work reports and discusses the potential of tissue culture 

technique as an alternative method for mass propagation and 

conservation of this interesting and attractive ornamental plant 

for future uses and exploitation. 

 

Index TermsIn vitro propagation, BAP, NAA, synthetic 

seeds. 

 

I.  I


NTRODUCTION

 

Oxalis triangularis, commonly known as “False 

Shamrock”, is an edible perennial plant belonging to the 

Oxalidaceae family. It is native to Brazil. Oxalis 



triangularis grow from bulbs, and their propagation is 

obtained by division of the bulbs. Like other bulbous plants, 



Oxalis triangularis goes through dormancy period on a 

regular basis. Somatic embryos and synthetic seeds 

(embryos or micro shoots encapsulated in artificial 

endosperms) hold great potential for large scale clonal 

propagation of superior genotypes of heterogeneous plants 

[1], [2]. They have also been used in commercial plant 

production and for the multiplication of parental genotypes 

in large scale hybrid seed production [3], [4].  

In Malaysia despite popular demand, this species is 

usually imported from Thailand, hence there is a necessity 

to mass propagate this species. Nowadays, mass propagation 

by using tissue culture method is quite common, the current 

work reports and discusses the production of artificial seeds 

from micro shoots derived from stem explants of this 

species. The subsequent in vitro regeneration was also 

achieved at high rates and the regenerated plantlets were 

successfully acclimatized. The development of encapsulated 

 

Manuscript received on March 5, 2013; revised May 6, 2013. This work 



was supported by University of Malaya Research Grants (RP025/2012A 

and PV025/2011B). 

All the authors are with the Institute of Biological Sciences, Faculty of 

Science, University of Malaya, 50603 Kuala Lumpur, Malaysia (email: 

rosna@um.edu.my, fara_aid@siswa.um.edu.my, jam_sya@yahoo.com, 

noorlidah@um.edu.my, mohajer.ae@gmail.com). 

or synthetic seed technology brings up a new prospect in 

agriculture and floriculture industry. Production of synthetic 

seeds is effective and acts as an important alternative 

method of propagation in commercially important plants, 

which do not produce seeds. Plants could be produced in 

large scale with high volumes. Consequently, genetic 

uniformity and stability of the plants could be maintained. 

Due to the sterility, it could be easily transported and has 

potential for long term storage without losing viability. The 

aims of this paper are to produce artificial seeds and to 

investigate the ability of the synthetic seeds to regenerate 

after 7 and 30 days of storage at low temperature (4 ± 1°C).   

 

II.  M


ATERIALS AND 

M

ETHODS



 

A.  Explant Sources and Encapsulation Matrix 

Micro shoots of Oxalis triangularis were induced from 

stem explants cultured on MS medium supplemented with 

0.5 mg/l NAA and 0.5 mg/l BAP. The stem cultures were 

maintained in the culture room at 25 

± 1 °C for 16 hours 

light and 8 hours dark. Illumination was at 1000 lux and 

relative humidity was 90 – 100%. The standard method for 

preparation of capsule matrix was followed [5]. 

B.  Final Stage Preparation of 3% (w/v) Sodium Alginate 

Solution (NaC

6

H

7

O

6

To prepare 3% (w/v) sodium alginate solution in 100 ml 

MS basal medium without calcium chloride dehydrate 

(CaCl


2

.2H


2

O), 1g sodium alginate powder was dissolved 

gradually. Sucrose (3.0 g) and hormones (1.0 mg/l NAA and 

1.5 mg/l BAP) were added. Media pH was adjusted to 5.8. 

This solution was autoclaved for 20 minutes at 121°C and 

104kpa. 


C.  Preparation of Calcium Chloride Dehydrate Solution 

(CaCl

2

.2H

2

O) 

Calcium chloride dehydrate solution was used as a 

complexion agent. To prepare 75 mM (w/v) calcium 

chloride dehydrate in 100 ml distilled water, 1.47 g 

CaCl

2

.2H



2

O was dissolved gradually. This solution was 

autoclaved for 20 minutes at 121°C and 104kpa.  

D.  Encapsulation Techniques and Bead Formation 

They were then, being dropped one by one in the 

CaCl

2

.2H



2

O solution. The micro shoots were allowed to get 

encapsulated (hardened) by allowing them to remain in 

CaCl


2

.2H


2

O solution for 30 minutes.  These beads were 

taken out and transferred into sterile distilled water to wash 

out the excess CaCl

2

.2H


2

O solution and were blotted with 

sterile tissue paper. 

Synthetic Seeds Production and Regeneration of Oxalis 



triangularis for Mass Propagation and Conservation 

Rosna Mat Taha, Noraini Mahmad, Jamilah Syafawati Yaacob, Noorlidah Abdullah, and Sadegh 

Mohajer 

International Journal of Environmental Science and Development, Vol. 4, No. 5, October 2013

461


DOI: 10.7763/IJESD.2013.V4.394

 

 

E.  Low Temperature Storage 

Sterile encapsulated beads of Oxalis triangularis were 

stored under dark condition at low temperature (4 ± 1°C). 

The survival rates for germination after 7 and 30 days were 

recorded. Thirty replicates were used in each treatment. 



F.  Data Analysis 

All experiments were conducted using a completely 

randomized design. Data collected were statistically 

analyzed using Duncan’s Multiple Range Test (DMRT). 

Mean with different letters in the same column differ 

significantly at p<0.05. 

 

III.  R


ESULTS

 

Synthetic seeds were created by encapsulation of   micro 



shoots of O. triangularis in sodium alginate solution (Fig. 

1a). The micro shoots were derived from stem explants of 

this species after being cultured for one month on MS 

medium supplemented with 0.5 mg/l NAA and 0.5 mg/l 

BAP. Table 1 shows the germination of synthetic seeds on 

MS basal media after 0, 7 and 30 days of storage. Even after 

30 days of storage, the artificial seeds retained the ability to 

germinate and gave high regeneration rate (90 %) and the 

percentage of survival rate was also high (77-86 %) as 

compared to control. Mean number of shoots formed was 

the highest after 7 days of storage. Root formation was 

observed in all treatments.  

 

TABLE


 

I:

 



G

ERMINATION OF 

S

YNTHETIC 



S

EEDS OF 


O

XALIS 

T

RIANGULARIS 

AFTER 


B

EING 


S

TORED AT 

D

IFFERENT 



D

URATIONS


 

Storage 


duration 

(Days) 


Conversion 

frequency 

(%) 

Percentage 



of survival

(%) 


No. of shoots 

(Mean ± SE) 

Root 

formation (%)



(control) 

60.00 

66.67 


2.10 ± 0.6a 

100.00 ± 0.0a

96.67 


86.21 

4.57 ± 0.7b 

100.00 ± 0.0a

30 


90.00 

77.78 


3.97 ± 0.7b 

100.00 ± 0.0a

Each value represents the mean ± SE of 30 replicates. The mean with 

different letters in the same column differ significantly at 0.05 level. 

 

TABLE


 

II:


 

S

YNTHETIC 



S

EEDS 


G

ERMINATION OF 



O

XALIS 

T

RIANGULARIS IN 

D

IFFERENT 



S

OWING 


S

UBSTRATES

 

Sowing 


substrate 

Treatment Observation 

MS basal 

medium 


Without hormone 

Encapsulated beads regenerated into 

complete plantlets. 

Black 


soil 

(non 


sterile) 

Moistened with 

distilled water 

Encapsulated beads started to shrink 

after a few days and eventually 

contaminated. 

Moistened with liquid 

MS without sucrose 

All encapsulated beads were 

contaminated with fungal infection.

Black 

soil 


(sterile) 

Moistened with 

distilled water 

Encapsulated beads showed no 

response and ultimately shrink. 

Moistened with liquid 

MS without sucrose 

All encapsulated beads were 

contaminated with fungal infection.

Each medium was represented by 20 replicates. 

 

Interestingly, synthetic seeds that were stored prior to 



being cultured showed significantly higher conversion 

frequency than synthetic seeds directly sowed on MS basal 

media (control). Higher percentage of survival was also 

recorded when the synthetic seeds were stored prior to being 

cultured on MS basal media compared to the control. It was 

also observed that storage period of 7 days was most 

optimum to ensure a high plantlet conversion rate and 

survival percentage. Shooting was also the highest from 

synthetic seeds stored for 7 days. This was clearly 

demonstrated in Table I.   

Table II indicates various media or substrates tested for 

germination of the synthetic seeds. The optimum substrate 

was MS basal medium (Fig. 1b and Fig. 2). After four 

weeks, the micro shoots elongated and eventually undergo 

complete regeneration. Other sowing substrates were shown 

to produce poorer results when compared to MS basal media. 

Substrates moistened with only distilled water resulted in 

shrinking of the synthetic seeds, while substrates moistened 

with liquid MS without sucrose resulted in fungal 

contamination. Finally the plantlets regenerated from 

synthetic seeds sowed on MS basal media were successfully 

acclimatized in the garden. 

 

 

Fig. 1. (a) Encapsulated beads of Oxalis triangularis before being stored at 



low temperature (4°C ± 1°C), (b) Shoots emerged from the encapsulated 

micro shoots of Oxalis triangularis after 7 days being cultured on MS basal 

medium. 

 

IV.  D



ISCUSSION

 

Creation and production of artificial seeds have been 



reported in many species [5], [6] - [9]. From Tables I and II, 

the observations after 7 days of encapsulated beads being 

germinated on the substrates both in black soil and sterile 

black soil gave negative results which failed to survive and 

finally the synthetic seeds died. On black soil substrate 

moistened with distilled water, the encapsulated beads 

during the first 7 days already showed shrinking sign where 

International Journal of Environmental Science and Development, Vol. 4, No. 5, October 2013

462


 

 

they failed to germinate and eventually contaminated with 



fungal infection. While synthetic seeds of Oxalis 

triangularis which were sown on sterile black soil 

moistened with distilled water did not show any sign of 

contamination, no germination response was observed. 

However, the beads eventually shrunk. Both synthetic seeds 

of  Oxalis triangularis sown in black soil and sterile black 

soil moistened with liquid MS without sucrose gave similar 

observations whereby, all encapsulated beads were 

contaminated with fungal infection after seven days. 

 

 

Fig. 2. (a) Development of micro shoots of Oxalis triangularis from 

synthetic seeds after 4 weeks being cultured on MS basal medium, (b) 

Regenerated plantlet of O. triangularis after successful acclimatization. 

 

Further studies were also carried out to compare synthetic 



seeds of Oxalis triangularis sown directly on MS basal 

medium, black soil and sterile black soil (Table II). The 

results showed that conversion frequency of synthetic seeds 

of  Oxalis triangularis into complete plantlets were better 

under  in vitro conditions compared to in vivo conditions. 

Synthetic seeds of Oxalis triangularis have the capability to 

respond when cultured on MS basal medium resulted in a 

high rate of plantlets regeneration. More than 60% 

successful regeneration from synthetic seeds of Oxalis 

triangularis was observed (Table II). Successful plant 

regeneration from synthetic seeds of Oxalis triangularis on 

MS basal medium was probably because the beads were 

cultured under aseptic conditions. Therefore, probability for 

the beads to get contaminated was much lower compared to 

seeds directly sown on in vivo substrates. 

The potential of synthetic seeds to be sown directly on in 

vivo substrates has received limited attention. From previous 

studies, Preece and West [10] were successful in sowing 



Hibiscus moscheutos directly into a greenhouse hydroponic 

system, both with and without light pretreatment. Bapat and 

Rao [11] conducted research on in vivo growth of 

encapsulated axillary buds of mulberry (Morusindica L.) 

and reported that addition of a fungicide to the alginate 

beads prevented contamination of the bud and increased 

survival of the buds when sown in soil. Ramakrishnappa [12] 

also observed that addition of fungicide (0.1% carbendazim) 

and bacteriocide (0.1% streptomycin) into the encapsulating 

gel reduced the incidence of fungal and bacterial 

contamination to a minimum level and nearly 95% of the 

beads remained healthy.  Researches need to be done to 

improve germination percentage of synthetic seeds when 

directly sown on non-sterile environment, such as in 

greenhouse or directly in the field. The present study proved 

that artificial seeds and tissue culture methods can be useful 

for plant multiplication and conservation. 

 

V.  C



ONCLUSION

 

In the present study, production of synthetic seeds was 



attempted from this species and the synthetic seeds managed 

to survive after 7 and 30 days after storage at 4°C. The rate 

of synthetic seeds conversion to complete plants after 7 days 

was 96.67% with 4.57 mean shoot numbers, while after 30 

days of storage, the conversion rate slightly decreased to 

90% with only 3.97 shoots formation per bead. 

A

CKNOWLEDGMENT



 

The authors would like to thank University of Malaya

Kuala Lumpur, Malaysia, for the Research Grant 

RP025/2012A and Postgraduate Research Grant 

PV025/2011B.  

R

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[2]  K. Mamiya and Y. Sakamoto, “A method to produce encapsulatable 

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Rosna Mat Taha was born in Malaysia on January 

22, 1958. In 1984, she obtained her B.Sc. in Botany 

from University of Malaya, Kuala Lumpur, Malaysia 

and eventually completed her Ph.D in 1989 on Plant 

Morphogenesis from University of Wales, Cardiff, 

UK. 

She is currently working as a lecturer (professor) at 



Institute of Biological Sciences, Faculty of Science, 

University of Malaya, Kuala Lumpur, Malaysia. She 

has published more than 100 papers in ISI-cited journals, proceedings, book 

chapters and etc. 

Prof. Taha is also an expert in plant tissue culture, morphogenesis, 

biotechnology, pigment and cellular behavior studies. She has won many 

awards (more than 20) in her research. She is a member of European 

Association for Reserach on Plant Breeding (EUCARPIA) and 

International Society for Horticultural Science  (ISHS). Also, she is the 

associate editor of Asian Journal of Biotechnology, Asian Journal of Plant 

Sciences and Journal of Applied Sciences. 

 

Noraini Mahmad was born in Kelantan, Malaysia on 

January 24, 1977. She obtained her B.Sc 

(Biotechnology) in 2000 from University of Malaya, 

Malaysia. Eventually in 2012, she obtained her M.Sc 

(Biotechnology) from University of Malaya, Malaysia. 

She is currently pursuing her Ph.D also at Institute of 

Biological Sciences, Faculty of Science, University of 

Malaya, Kuala Lumpur, Malaysia. 

To date, she had authored 2 scientific articles 

published in ISI-cited journals. She also had participated in several 

conferences such as 1

st

 International Symposium on Sustainable Vegetable 



Production in South East Asia (2011). 

Jamilah Syafawati Yaacob had obtained her B.Sc 

(Biotechnology & Biochemistry and Molecular 

Biology) in 2007 from University of Melbourne, 

Australia and eventually her M.Sc (Crop 

Biotechnology) in 2009 from the University of 

Nottingham Malaysia Campus. She had completed 

her Ph.D in early 2013 on Plant/Crop 

Biotechnology from University of Malaya, 

Malaysia. 

Upon completing her Ph.D, she is currently serving University of Malaya 

as a senior lecturer at the  Institute of Biological Sciences,  Faculty of 

Science, University of Malaya, Kuala Lumpur, Malaysia. To date, she had 

published 10 articles in reputable ISI-cited journals and proceedings. She 

also had participated and won awards in various conferences and 

exhibitions related to her work. 

 

Noorlidah Abdullah had graduated in B.Sc (Medical 

Biochemistry) in 1984 from University of Surrey, 

United Kingdom, M.Sc (Mycology) in 1989 and Ph.D 

(Applied Mycology) in 1997 both from University of 

Malaya, Malaysia.   

     She  has  contributed  substantially  to  research, 

teaching and administration since she joined the 

Institute of Biological Sciences in 1997. Later she was 

promoted to Associate Professor in Oct 2002 and then 

professor in 2008 and has become well known for her 

research in Fungal Biotechnology and Food Mycology. 

    She  was  given  due  recognition  for her expertise in food mycology 

(mycotoxins) by the Ministry of Health for whom she has been an expert 

committee in Asean Expert Group On Food Safety and Food Additives and 

Contaminants Committee. She has published 70 articles in reputable 

journals and has supervised 48 postgraduate students.  

 

Sadegh Mohajer had obtained his M.Sc (Plant 

Breeding) in 2008 from Azad University, Iran and 

currently pursuing his Ph.D in Plant Biotechnology at 

Institute of Biological Sciences, Faculty of Science, 

University of Malaya, Kuala Lumpur, Malaysia. His 

Ph.D is currently being sponsored by the university 

under the Bright Sparks scholarship.  

Prior to joining the University of Malaya, Malaysia, 

he had worked as a research scientist at Iran-

Levasanat Agricultural Extension Center until 2010 

and had also given lectures for Experimental Design Course at Payamnur 

University (2009-2010). To date, he had authored over 12 articles, edited 1 

book and 2 book chapters. He also had taken part in more than 5 

international conferences and won several awards, such as the best science 

expert at Iran-Shemiranat Agricultural Organization in 2010. He was also 

ranked first in Iran Plant Breeding M.Sc entrance examination in 2006.  

Furthermore, he was also among the editorial board members of Journal 

of Sciencia and had evaluated scientific articles for African Journal of 

Biotechnology and Scientia Horticulturae. 



 

 

International Journal of Environmental Science and Development, Vol. 4, No. 5, October 2013



464


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