PHILIPP AGRIC SCIENTIST ISSN 0031-7454

Vol. 103 No. 3, 245-255

September 2020

Research Note

In Vitro Seedling Growth of Adlay (Coix lacryma-jobi L.) ‘Pulot’

and its Response to 2,4-Dichlorophenoxyacetic Acid,
Benzylaminopurine, Plain Cow Dung Ash and Agnihotra Ash
Emmanuel L. Bernardo1,*, Pamela G. Fernandez1, Evalour T. Aspuria1, Nina M. Cadiz2
and Rocky T. Marcelino3
1
Institute of Crop Science, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna,
Philippines
2
Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños, College, Laguna,
Philippines
3
Institute of Statistics, College of Arts and Sciences, University of the Philippines Los Baños, College, Laguna, Philippines
*
Author for correspondence; e-mail: elbernardo@up.edu.ph

Adlay (Coix lacryma-jobi L.) is an emerging alternative cereal crop which may benefit from plant tissue
culture approaches. In this study, the morphogenic response of mature embryos of adlay to 2,4-
dichlorophenoxyacetic acid (2,4-D; 1–3 ppm) and benzylaminopurine (BAP; 1–3 ppm) in Murashige and
Skoog (MS) basal medium was screened in vitro. Further, agnihotra ash (AA) and cow dung ash (CDA) were
evaluated as unconventional culture media additives. A sterilization procedure was first developed for
mature embryos of adlay by varying sodium hypochlorite (NaOCl) concentration and the duration of surface
sterilization. NaOCl at 1% (v/v, Chlorox®) using double sterilization (7 min: 8 min) was effective for surface
disinfection of dehusked adlay seeds. In terms of plant growth regulators (PGRs), BAP and 2,4-D enhanced
shoot growth in lower doses, but primary root growth was inhibited. Retarded shoot and root development
were observed even at the lowest 2,4-D concentration and as BAP was increased. Yellow, compact callus
was observed surrounding the mesocotyl and white crystalline and loose callus around the radicle region.
Both types of calli appeared non-embryogenic and were most frequent at 2 and 3 ppm 2,4-D regardless of
BAP concentration and ash additives. As for ash additives, application of 0.01% cow dung ash, 0.01%
agnihotra ash and 1% cow dung ash resulted in larger seedlings compared with 1% agnihotra ash in basal
media, although these differences were not pronounced in the plant growth regulator (PGR) background.
Agnihotra ash and cow dung ash as miscellaneous additives may provide cost savings in plant tissue
culture; further work is warranted.

Key Words: adlay, agnihotra ash, Coix lacryma-jobi L., cow dung ash, in vitro seedling growth, plant tissue culture
Abbreviations: AA – agnihotra ash, BAP – benzylaminopurine, CDA – cow dung ash, CIM – callus induction medium,
NaOCl – sodium hypochlorite, PGR – plant growth regulator

nutrients—may soon threaten the advantages of tissue

INTRODUCTION
In plant tissue culture, as in the natural environment,
mineral salts support growth, whereas plant growth
regulators direct growth and morphogenesis. In some
cases, manipulating the proportion of nutrient salts alone
can also regulate and control morphogenesis in vitro
(Niedz and Evens 2007). These and other culture medium
components are the primary determinants of the cost
efficiency of propagating plants by tissue culture.
However, unabated increases in the price of some
components—basic chemicals used as sources of essential
culture. Agar is estimated to contribute 70% of the cost of
the medium, and salts, hormones, and other additives
only 5% to 15% (Prakash et al. 2004). While using
alternatives for the gelling agent (Prakash et al. 2004) and
eliminating the use of sugar (Kozai et al. 2005) can help
keep tissue culture costs low, the mineral salts and plant
growth regulators have fewer substitutes. Moreover,
Murashige and Skoog (MS) basal medium (1962), despite
being the basic and universal tissue culture medium,
cannot supply the distinct requirements of other plants
for nutrition and many other plant species benefit from

The Philippine Agricultural Scientist Vol. 103 No. 3 (September 2020)

 In Vitro Seedling Growth of Adlay Emmanuel L. Bernardo et al.

deviations from the original recipe. Hence, an array of marginal soil where other crops may not thrive, and does
tissue culture media has been formulated to suit the not require much attention. Adlay is also nutritious,
diverse nutritional requirements of plants in vitro (George having higher protein and fat contents than any of the
et al. 2008). more popular cereal grains (Van den Bergh and
Iamsupasit 1996). Renewed interest in adlay in the
Minor changes in the nutritional composition of the
Philippines has resulted from increasing pressures in
medium can direct or alter growth without the necessity
production and rapid conversion of prime agricultural
of growth regulators (Niedz and Evens 2007; Ramage and
lands to commercial use. Beyond its potential as a cereal
Williams 2002). Alternative components that may
substitute, adlay offers medicinal properties that warrant
complement these chemical compounds as sources of
consideration (Lee et al. 2008; Wang et al. 2012), and this
plant nutrition in vitro are essential to reduce the
attribute can be exploited through plant tissue culture
technology’s cost. This should also facilitate plant
systems.
propagation through tissue culture to be resource
effective. The main objective of the study was to evaluate the in
vitro response of adlay mature embryos to 2,4-
Except for amino acids and vitamins, organic growth
dichlorophenoxyacetic acid (2,4-D) and
additives (e.g., aloe vera gel) incorporated into the
benzylaminopurine (BAP) to provide the basis for future
medium are uncommon. Some ‚miscellaneous‛ or
tissue culture studies. Basal MS with BAP was used to
undefined additions can help by providing growth-
screen for levels suitable for micropropagation while
promoting factors (George et al. 2008). For instance,
combinations of 2,4-D and BAP were used for callus
locally, banana homogenate and tomato paste have found
induction for the plant transformation experiments.
unconventional use in orchid tissue culture. In banana
Further, the response of the embryo to steeped agnihotra
and abaca micropropagation, 10% to 15% (v/v) young
ash and cow dung ash was explored to determine
coconut water is routinely used in the multiplication
whether or not ash would have some beneficial effect
phase. Indeed, coconut water promotes growth among
when added to culture media.
many other species (Peixe et al. 2007). Such organic
additions produce plant responses that are difficult to MATERIALS AND METHODS
predict, but over the years and by experience, this
unpredictability has been within acceptable bounds. Experiments were conducted at the Plant Tissue Culture
Laboratory, Room C-211, Institute of Biological Sciences,
Activated carbon is a popular additive because of its University of the Philippines Los Baños (UPLB), College,
wide application in tissue culture. Conventionally Laguna, Philippines from January 2014 to February 2014.
considered inert, its role is to adsorb toxic substances,
indirectly regulate exogenous hormones and promote Plant Material
better growth (Thomas 2008). Similar physical functions
Dry and mature seeds of adlay cv. Pulot were obtained
may be undertaken by the ash derived from plant
from field grown plants of the Southern Tagalog
cellulosic materials. Agnihotra ash increased callus fresh
Integrated Agricultural Research Center (STIARC) of the
weight in carrot by up to 72% (Bernardo and Aspuria
Department of Agriculture in Lipa City, Batangas,
2012). This ash is produced by the burning of organic
Philippines. The seeds were harvested in October 2012.
materials in a copper pyramid (Deshpande and Potdar
The seeds were approximately 1-yr-old when studied.
1998). An x-ray diffraction analysis reveals that the ash
Dry and mature seeds of adlay were completely dehulled
contains four macronutrients and five micronutrients
by hand, avoiding damage to any of the internal
along with some trace elements (Fernandez 2007). This
structures.
inexpensive resource provides several demonstrated
benefits in agriculture (Fernandez 2007; Berk and Johnson Experiment I
2009). Studying the compatibility of agnihotra ash and
Preparation of seed explant and sterilization procedure
tissue culture could produce some insights into mineral
nutrition and other unidentified factors that may Seeds were washed in detergent solution and rinsed
influence plant growth in vitro. under running water for 15 min. Pre-washed seeds were
subjected to different sterilization treatments, then three
A lesser known crop that is gaining interest as an
to six thorough rinsings in sterile, distilled water. The
alternative staple food is adlay (Coix lacryma-jobi L.)
factor and factor levels were NaOCl concentration and
(Schaaffhausen 1952). Unlike rice and corn, it is less
duration of double sterilization at 1.0%, 1.5%, and 2.0%
susceptible to pests and diseases, grows well in poor or
(v/v) and 7 min:8 min and 10 min:10 min sequences,

246 The Philippine Agricultural Scientist Vol. 103 No. 3 (September 2020)
In Vitro Seedling Growth of Adlay Emmanuel L. Bernardo et al.

Table 1. Relative abundance of detectable elements in cow

dung cake and agnihotra ash as percentages by weight. Experiment II
Analysis was performed by BIOTECH, University of the
Philippines Los Baños via x-ray diffraction and fluores- Isolation and culture mode of the embryos
cence analyses.
Cow Dung Cake Agnihotra Ash The explant was the zygotic embryo obtained from
Elemental Atomic Elemental Atomic mature dry dehulled seeds of adlay. Seeds were surface-
Calauan, Laguna, Philippines sterilized using 1% NaOCl following the double
C 41.45 49.04 42.13 54.02 sterilization (7 min: 8 min) procedure as in Experiment 1.
O 55.52 49.32 40.04 38.54
The seeds were then soaked in sterile distilled water for
Mg 1.42 0.83 0.60 0.38
Si 1.62 0.82 - -
approximately 24 h to soften the seeds for ease of embryo
K - - 11.41 4.49 extraction. Before extraction, presoaked seeds were
Ca - - 1.42 0.55 resterilized with 20 mL 1% NaOCl (v/v) for 10 min.
P - - 1.50 0.74 Thereafter, the seeds were rinsed three to five times with
S - - 1.02 0.49 sterile distilled water. Following the procedure on barley
Cl - - 1.67 0.73 by Sharma et al. (2005), mature embryos were excised
Mn - - 0.22 0.06 from the endosperm with the aid of a sharp scalpel.
Olango Island, Lapu-Lapu City, Philippines
Embryos were inoculated with embryonic axis-side down
C 38.98 46.94 9.77 15.87
O 56.34 50.95 47.06 57.39
and in full contact with the different MS-based media
Mg - - 1.89 1.51 formulations.
Si 2.72 1.40 21.10 14.65
Preparation of ash infusions
K - - 5.14 2.57
Ca 1.96 0.71 7.10 3.46 Agnihotra ash (AA) was obtained from Dr. Pamela G.
P - - 3.28 2.07 Fernandez, using methods as prescribed for the agnihotra
Cl - - 1.8 0.99 process (Bizberg 1999). Cow dung ash (CDA) was
Al - - 1.32 0.91
produced by burning dried cow dung patties. The
Fe - - 1.54 0.54
composition of AA and CDA is shown in Tables 1 and 2.
A 1,000 ppm stock ash infusion was prepared by steeping
respectively (Table 1). Four seeds in a Petri dish (90 x 1 g agnihotra ash or cow dung ash in 1000 mL sterile
15 mm) served as the experimental unit. Each treatment distilled water for 3 d (Koch 2004).
was replicated 12 times. Disinfection was performed
Culture medium and growth conditions
inside a horizontal flow laminar chamber. Surface-
sterilized seeds were inoculated onto 20-mL Murashige Each test tube (9 x 20 mm) was filled with 10 mL MS
and Skoog’s (MS) basal medium (1962) in 90 x 15 mm media supplemented with 2,4-D (0, 1, 2, 3 ppm), BAP (0,
Petri dishes and routinely monitored (2, 4, 7 d after 1, 2, 3 ppm), agnihotra (0, 0.01, 1%) or cow dung (0, 0.01,
inoculation) for extent of contamination (in percentage). 1%) ash infusions. The pH was adjusted to pH 5.8 before
Actual counting of normally germinated seeds was done adding 5.8 g L-1 of Pronadisa® PTC Agar and autoclaving
on day 7. A seedling was considered ‘normal’ if all at 121°C (15 psi) for 15 min. The test tubes were then
seedling parts (coleoptile, plumule, radicle, and covered with cotton plugs and sterile paper secured with
coleorhizae) were present and met standards for normal elastic bands. Adlay cultures were maintained in a culture
(ISTA 1996) with no apparent damage (e.g., necrotic parts
due to oxidative injury) to any tissue part caused by Table 2. Chemical composition of cow dung ash (Adapted
sterilization. from Omoniyi et al. 2014).
Oxide Weight (%)
Data collection and experimental design and analysis
S iO 2 69.65
The sterilization experiment was designed as a three by Al2O3 4.27
two (3 x 2) factorial experiment in CRD. Fe2O3 2.99
CaO 12.55
The number of non-contaminated seedlings, coleoptile MgO 2.22
emergence, radicle emergence, and true leaf growth were SO3 1.36
recorded. The data (in %) were processed using arcsin K2O 2.94
transformation and analyzed using two-way ANOVA Na2O 0.57
with the aid of Statistical Analysis System Software v.9.3. P2O5 1.48
Treatment means were compared using Fisher’s least Mn2O5 0.63
significant difference (LSD) at α=0.05. T iO 2 0.33

The Philippine Agricultural Scientist Vol. 103 No. 3 (September 2020) 247
 In Vitro Seedling Growth of Adlay
room at ambient temperature of 25°C + 2°C under
approximately 40 µmol m-2 s-1 of continuous white light
supplied (24 h) by cool white fluorescent lamps (Philipps,
40 W) for 15 d.
Experimental design and data analysis
The experiment was designed as a 4 x 4 x 6 full factorial
experiment in CRD. A test tube (180 x 20 mm) containing
a single explant served as an experimental unit. Each
treatment comprised 10 test tubes which served as
replicates. Three trials were performed. However, the first
and second trials succumbed to very high contamination
rates, mostly bacterial, and had very few samples to be
adequately analyzed. Only the third trial had enough
samples to permit a proper statistical analysis.
The data were analyzed using two-way ANOVA with
the aid of Statistical Analysis System Software v.9.3. Post-
hoc analyses of significant effects were done by doing
group comparison using linear orthogonal contrasts.
Group means were compared as follows: Agnihotra ash
vs. cow dung ash, with BAP vs. without BAP, 0.01%
agnihotra ash vs. 1% agnihotra ash, and 0.01% cow dung
ash vs. 1% cow dung ash.
RESULTS AND DISCUSSION
Response of Mature Embryo Explants to Surface
Sterilization
Mature embryos as explants for callus induction and
multiple shoot formation are not uncommon, especially
in grasses. Some examples from Poaceae include maize
(Zhao CH et al. 2010), sorghum (Zhao L et al. 2010) and
wheat (Sarker and Biswas 2002; Malik et al. 2004) and rice
(Noor et al. 2011). Even in model grass Brachypodium
distachyon, mature embryos are compatible systems for
callus and plant regeneration studies (Yu et al. 2019).
Immature embryos are also ideal explants because of
their high regenerative capacities, but their small size
makes them difficult to extract from a developing seed
compared with a mature embryo that can be excised
using the naked eye. A third important reason is that
mature seeds are more available throughout the year
unlike immature embryos which are only available
during a limited time window in a cropping cycle.
For adlay, a surface sterilization protocol is virtually
non-existent. The goal of surface sterilization is not only
to eliminate contaminants but also to ensure that plant
growth will continue under in vitro conditions.
Contamination-free cultures are of no practical use if the
explants had died from chemical injury because of
excessive sterilization. Therefore, deciding what
248 The Philippine Agricultural Scientist Vol. 103 No. 3 (September 2020)
Emmanuel L. Bernardo et al.
concentration to use and the extent of contact will largely
depend on its effect on the tissue or the explant’s
regeneration potential.
The various concentrations of NaOCl and the
duration of disinfection did not differ in terms of degree
of contamination (p=0.584) and radicle growth (p=0.472)
(Table 3). Further, no significant differences were detected
in the main effects of either NaOCl concentration
(p=0.691) and duration (p=0.866). On average, three out of
four seedlings were contamination-free per Petri dish
regardless of NaOCl concentration or duration of contact.
A 1% NaOCl concentration was enough to control
external contaminants, while increasing the concentration
up to 2% had no added benefit. Similarly, shorter
disinfection duration (7:8 min) was as effective as the
longer disinfection period (10:10 min). On the other hand,
the two durations that were not different in terms of the
level of contamination gave some difference in terms of
radicle emergence; the shorter duration gave better
radicle emergence (p=0.049) (Table 3).
The concentration and duration of disinfection
treatments resulted in significant differences in
emergence of the first true leaf (p=0.006) (Fig. 1). The
emergence of the coleorhiza/radicle, which signals the
onset of germination, occurred approximately 24 h after
inoculation. After 2 or 3 d, the coleoptile, and
subsequently the first leaf, also emerged and showed no
apparent sign of chemical injury. The concentration and
duration of sterilization can lower the number of normal
seedlings, i. e., with intact and functional shoots and
roots. The failure of embryo explants to develop either
root or shoot or both structures seemed to be an outcome
of chlorine toxicity, as was the case in six peanut varieties
(Maina et al. 2010) where regeneration was largely
determined by the disinfection treatment and to some
extent, by genotype. The 1% NaOCl concentration is also
effective in Achyranthes aspera seed explants (Sen et al.
2014) with no adverse effect on germination and growth.
Chlorine toxicity or oxidative damage is more likely in
Table 3. Contamination-free explants and radicle emer-
gence of adlay mature embryo explants in vitro.
Contamination-free Radicle Emergence
(%) (%)
Duration (min)
7:8 93.75 a 58.33 a
10:10 94.44 a 43.06 b
Concentration (%, v/v)
1.0 91.67 b 53.13 b
1.5 95.83 b 52.08 b
2.0 94.79 b 46.88 b
Means followed by different letters in a column for each dura-
tion and concentration are statistically significant using LSD at
α = 0.05.
In Vitro Seedling Growth of Adlay Emmanuel L. Bernardo et al.

shoots via indirect organogenesis, a morphogenic

pathway exploited in plant transformation experiments.
The effect of 2,4-D on seedling growth was to retard
primary root elongation and thus only shoot lengths
could be measured. The analysis (Table 4) showed the
absence of an interaction between BAP and ash additives
(p=0.120). Main effects were highly significant for both
BAP (p<0.0001) and ash additives (p=0.002).

Seedling growth was greatly altered if only 2,4-D was

present in the medium. Increasing 2,4-D concentrations to
2 ppm and 3 ppm resulted in shorter shoots (Fig. 3), the
complete inhibition of primary and lateral roots and
prominent morphological abnormalities. In this set of
treatments, the shoots were short and acutely bent, while
Fig. 1. Interaction plot of different durations of soaking x
root development was completely inhibited. It seemed
concentration of NaOCl in terms of emergence of first true
leaf. The number of emerged leaves was counted on the 7th that the coleoptile failed to rupture on one side, which
d after inoculation. restrained the vertical growth of the shoot and resulted in
the characteristic bending or an undulating growth
adlay considering that the dehulled embryo is
pattern. The ‚tensile skin theory‛ (Kutschera and Niklas
unprotected and permeable. Mishaps in excision
2007), which supports auxin-induced stimulation of the
procedure that caused mechanical injury were also
extensibility of the inner sub-epidermal cells, is likely
possible.
operative but possibly perturbed because of exogenously
In adlay, 1% NaOCl at the shorter duration (7:8 min) supplied 2,4-D.
was adequate to prevent a high contamination rate for
Irregularities in primary root growth were thought at
embryo explants. A high survival rate in vitro for adlay
first as the early signs of callus development. However,
was expected since the seed and thus the embryo was
closer inspection under high magnification revealed an
protected by the tight hull.
enlarged mesocotyl and suppressed root growth. Such
Morphogenic Response to Basal Media morpho-physiological disorders are not uncommon in
tissue culture where not only medium composition affects
The first visible response was the emergence of the
growth but also the immediate environment inside the
coleorhiza, followed by the radicle within the first 24 h.
vessel (Kozai et al. 2005; Hazaraki 2006). However, the
Three days after inoculation, the coleoptile and first true
relatively short culture duration (15 d) points to the
leaf emerged as was observed in Experiment I. Seedlings
greater influence of 2,4-D on shoot growth rather than
growing on the basal medium (no 2,4-D, BAP or ash
that of environment factors in vitro. Roots are particularly
additives) were robust. Primary root and shoot elongation
sensitive to auxin (Radermacher and Klambt 1993), and
were noticeably ahead than those with either 2,4-D or
BAP in the medium. Table 4. Chemical composition of cow dung ash (Adapted
from Omoniyi et al. 2014).
The plant growth regulators (PGRs) affected growth
Mean
differently (Fig. 2); there was a strong three-way Source of Variation DF Pr>F
Squares
interaction (p<0.0001) on shoot growth among 2,4-D, BAP, Treatment 19
and ash additives (data not shown). To allow for a more BAP (A) 3 99159.4763 <.0001
insightful interpretation, the 2,4-D group (Table 4) was Ash (B) 4 9049.5709 0.0018
analyzed independently of the treatments with BAP AA vs. CDA (1) 6563.4141 0.0757
(Table 6) and discussed separately in the following With BAP vs. no BAP (1) 284187.4863 <.0001
sections. AA 0.01% vs. AA 1% (1) 2947.2892 0.2334
CDA 0.01% vs. CDA 1% (1) 7459.4911 0.0583
Morphogenic Responses to 2,4-D, BAP and Ash
AB 12 3053.3620 0.1303
The response of embryo explants to 2,4-D along with BAP Error 406 2069.276
and ash was evaluated to explore the possibility of using Total 425
mature embryos for callus induction in adlay. The callus CV (%) 60.74
phase serves as a transitory role to produce multiple BAP – 6-benzylaminopurine
AA – Agnihotra ash
CDA – cow dung ash

The Philippine Agricultural Scientist Vol. 103 No. 3 (September 2020) 249
 In Vitro Seedling Growth of Adlay Emmanuel L. Bernardo et al.

this sensitivity is Table 5. Frequency (%) of callus-like formation in the

mesocotyl region of adlay inoculated onto MS basal me-
enhanced in the dium supplemented with 2,4-D and BAP and steeped
presence of ethylene agnihotra or cow dung ash.
(Park et al. 2011) that FREQUENCY OF CALLUS-LIKE FORMATION (%)
accumulates quickly 2,4-D 0 1 2 3
in vitro. A strong BAP
repressing effect of 0 0 0 0 0
2,4-D on root growth 1 100 100 0 0
has been reported in 2 100 0 100 100
3 89 90 0 0
maize seedlings
BAP + 0.01% Agnihotra Ash
(Martinez-de la Cruz
0 0 0 0 0
et al. 2015), and the
1 100 11 0 0
root inhibition in 2 89 83 10 25
adlay is probably of 3 89 67 0 67
the same mechanism. BAP + 1% Agnihotra Ash
0 0 0 0 0
Typical in treatments
1 0 0 42 0
with 2,4-D was the
2 86 10 0 67
development of
3 100 100 100 0
yellow-green, BAP + 0.01% Cow Dung Ash
compact structures 0 0 0 0 0
surrounding an 1 71 0 0 0
enlarged mesocotyl, 2 100 88 88 50
or sometimes in the 3 88 100 100 75
root region (Fig. 4A). BAP + 1% Cow Dung Ash
White, crystalline, 0 0 0 0 0
and loose structures 1 70 20 30 10
also formed around 2 80 70 70 80
3 100 70 50 30
the root-embryo
region (Fig. 4B). The
frequency of
supports callus initiation and proliferation.
occurrence of callus-
like formation in the BAP promoted longer shoot growth than the control
mesocotyl region is in conjunction with 1 ppm 2,4-D and highlights the
shown in Table 5. known role of cytokinins in stimulating growth together
Callus-like growth with auxin by promoting cell division. Shoot length
was recurrent at 3 tended to increase as the level of BAP reached 3 ppm,
ppm 2,4-D with 1% although 2,4-D seemed to have an overriding effect as
agnihotra ash or group averages became shorter as 2,4-D increased. In
Fig. 2. Adlay seedlings growing 0.01% cow dung ash conjunction with an auxin, BAP controls primary root and
in MS basal medium with 2,4-D but was less frequent lateral root formation in Arabidopsis (Muraro et al. 2013).
and BAP. Bar = 5 cm.
in 1% cow dung ash. In this study, however, adding only BAP to the medium
The initiation and did not provide any benefit in terms of seedling growth
development of callus-like growth in the mesocotyl and since roots were inhibited, similar to treatments with 2,4-
root regions of adlay followed a similar fashion as in two D only. In rice, kinetin and zeatin were found to inhibit
sweet sorghum varieties, ‘Yuantian No. 1’ and ‘M81E,’ the initiation of lateral roots but stimulated lateral root
wherein the swelling of the mesocotyl and radicle regions elongation observed within 10 d after a pre-germination
was the first visible mark of primary callus initiation in step (Rani Debi et al. 2005). The in vitro response of adlay
germinating seeds in 2,4-D-containing media (Zhao L et corroborated this finding.
al. 2010). Unlike naturally-occurring auxins, 2,4-D is not
Morphogenic Responses to BAP and Ash
transported out of the cell (Delbarre et al. 1996) and is
poorly metabolized (Hošek et al. 2012), which allows A set of BAP treatments were tested to screen for multiple
accumulation inside the cells (Motte et al. 2014) and shoot formation that can be useful in micropropagation

250 The Philippine Agricultural Scientist Vol. 103 No. 3 (September 2020)
In Vitro Seedling Growth of Adlay
Fig. 3. Shoot length of adlay. in MS-based medium supplemented with 2,4-D and 1–
3 ppm BAP and steeped Agnihotra ash (AA) and cow dung ash (CDA), (v/v).
Vertical bars represent the s.e. (n = 8–10). Average line = 48.5 mm. Some data are
missing due to contamination.
and plant regeneration studies in adlay. A combined
effect was present between BAP and the ash additives
(Table 6) in terms of shoot (p=0.0002), mesocotyl
(p<0.0001), and root lengths (p<0.0001) but absent in terms
of the number of roots formed (p=0.563). All the basic
parts of a normal seedling can be identified, although the
size of each organ varied and was affected by either BAP
or ash additives.
Shoots in BAP were phenotypically similar to normal
seedlings grown in basal MS medium (Fig. 2, topmost
panel). Primary root elongation and lateral root formation
was significantly reduced in treatments with BAP.
Increasing the level of BAP did not affect normal shoot
The Philippine Agricultural Scientist Vol. 103 No. 3 (September 2020)
Emmanuel L. Bernardo et al.
development or primary root count
but had a retarding effect on further
shoot growth and lateral root
formation, which underpins the
classical role of cytokinin involving
the shoot apical meristem (Bishopp
2011) alongside its reverse effects
on the root apical meristem (Azizi
et al. 2015). Cytokinin antagonizes
auxin in many processes, which
include lateral root development
and root architecture (Lavenus et
al. 2013).
Shoot and root growth varied
according to ash treatment (Fig. 5).
Cow dung ash promoted longer
shoot growth although longer roots
were stimulated by agnihotra ash.
Overall, larger seedlings resulted
from the use of cow dung ash,
although the difference with the
control was small. Agnihotra ash
gave smaller seedlings. The 0.01%
concentration was generally growth
-promotive and at the same efficacy
range of most PGRs when used to
manipulate plant growth in vitro.
When incorporated directly into the
callus induction medium (CIM),
0.01% agnihotra ash produced
larger carrot callus in both primary
and secondary subcultures
compared to CIM without
agnihotra ash (Bernardo and
Aspuria 2012). Germination studies
also showed that agnihotra ash was
more potent at a lower dose
(Angeles 2008; Cammayo 2008).
At 1% agnihotra ash, root growth, and to some extent
mesocotyl, were clearly much shorter than the rest of the
treatments. There are indications that differential growth
in adlay among ash types and their respective
concentrations may be a function of the composition of
the ash additives. Extracts of cow dung ash, obtained by
dissolving the ash and evaporating most of the water
until crystals started to form, showed that K, Na, and Mg
occurred in the highest concentration, followed by Ca, Fe,
Al, and Zn (Waziri and Suleiman 2013). An earlier
elemental analysis of agnihotra ash, on the other hand,
showed a similar constitution but differing in the relative
proportions of elements (highest in K and Ca, by weight)
(Fernandez 2007). Mineral composition could also vary by
251
 In Vitro Seedling Growth of Adlay Emmanuel L. Bernardo et al.

Table 6. Analysis of variance of shoot, mesocotyl, and root lengths of adlay embryos inoculated onto MS basal medium
supplemented with BAP and ash additives at 15 d after inoculation.
SHOOT LENGTH MESOCOTYL LENGTH ROOT LENGTH
Source of Variation DF Mean DF Mean DF Mean
Pr>F Pr>F Pr>F
Squares Squares Squares
Treatment 19 19 19
BAP (A) 3 14363.08742 <.0001 3 65.2518489 0.0067 3 4051.46662 <.0001
Ash (B) 4 4262.76062 <.0001 4 186.6394239 <.0001 4 835.29209 0.0011
AA vs. CDA (1) 13143.54628 <.0001 (1) 456.2181772 <.0001 (1) 0.25310 0.9693
With BAP vs. no BAP (1) 41089.24157 <.0001 (1) 5.7695414 0.5410 (1) 11217.69291 <.0001
AA 0.01% vs. AA 1% (1) 1801.05019 0.0272 (1) 36.3423818 0.1265 (1) 2724.15718 0.0001
CDA 0.01% vs. CDA 1% (1) 1675.66291 0.0331 (1) 2.0495835 0.7155 (1) 21.58541 0.7228
AB 12 1227.99665 0.0002 12 73.3334162 <.0001 12 880.43951 <.0001
Error 128 360.9562 127 15.357996 119 170.73855
Total 147 146 138
CV (%) 18.56 53.94 38.00
BAP – 6-benzylaminopurine
AA – Agnihotra ash
CDA – cow dung ash

location (Table 1), presumably because of variation in the
cow’s diet and physiology. The difference in ash
compositions may explain the differences in adlay
growth between the two ash types.
SUMMARY AND CONCLUSION
Using adlay as an experimental crop, two independent
experiments were designed to determine a suitable
surface sterilization procedure for Coix lacryma-jobi L. and
assess the morphogenic response of excised mature adlay
embryos in vitro with selected combinations of 2,4-D and
BAP, steeped agnihotra ash and steeped cow dung ash
solutions as a basis for future tissue culture studies on
adlay. Indeed, tissue culture of adlay and the application
of unconventional additives in minute amounts for
growth enhancement are pioneering studies. A similar
preliminary study was conducted on carrot (Bernardo
and Aspuria 2012) and the present research serves as
follow up.
The most effective double sterilization procedure for
adlay is using 1% NaOCl (v/v, Chlorox®, 5.25% a. i.) for
Fig. 4. A) Yellow to yellow-green compact callus-like
formations (*) around the mesocotyl region of adlay in
treatments with 2,4-D. Bar = 1 mm. B) Acute bending,
suppressed nodal root growth and formation of white,
crystalline callus in the root-embryo axis in 2,4-D
supplemented media. Bar = 1 mm.
15 min. A higher concentration and longer duration can
be detrimental to radicle growth and leaf emergence.
From inoculated adlay embryos, normal shoot
growth was obtained in MS basal medium without PGRs.
Although lateral root formation was vigorous in basal
medium, it was inhibited in 2,4-D and BAP containing
medium applied singly, or in combination. The response
of adlay embryos in the basal medium and ash additives
were more consistent in treatments without 2,4-D
suggesting an overriding effect by 2,4-D. Longest shoot
growth was obtained in 1 ppm 2,4-D and BAP. Increasing
the concentration of 2,4-D beyond 2 ppm in the absence
of BAP resulted in reduced shoot growth and the
inhibition of radicle emergence. BAP, applied singly, at 1
ppm, resulted in plants with longer shoots but without
fully developed roots. Increasing the concentration of
BAP did not result in multiple shoot induction.
Moreover, friable and embryogenic callus was not
observed in the set of PGRs tested.
Compared with the control, ash additives had a
growth-promotive effect. Agnihotra ash (AA) and plain
cow dung ash (CDA) at 0.01% were stimulatory to shoot
and root growth in tissue culture. Primary root length
was particularly stimulated at 0.01% AA. A trend was
that 1% AA ash gave smaller seedlings, even in the
absence of 2,4-D or BAP, which suggested that it was not
beneficial to shoot and root growth. The reason for this
negative effect on growth is not yet known. The
mechanism for the growth-promotive effects of agnihotra
or cow dung ash in adlay in vitro is currently unclear.
Unlike PGRs with a defined series of transport, signal
perception, transduction, and expression regulated by a
subset of genes, the mode of action of cow dung ash and
agnihotra ash may be entirely physical. When ash is
added to water, a hypothesis is that a portion of the ash
dissolves and a fraction can remain suspended in the

252 The Philippine Agricultural Scientist Vol. 103 No. 3 (September 2020)
In Vitro Seedling Growth of Adlay
Fig. 5. Overall plant growth of adlay in MS-based medium
with BAP (in ppm) and steeped agnihotra ash (AA) and
cow dung ash (CDA), (v/v). Shoot, mesocotyl, and root
lengths were measured 15 d after inoculation. Vertical
bars represent the s.e. (n = 8–10). Group average line =
103 mm.
medium in colloidal form; because of their small size and
large surface area, suspended particles could have an
effect on nutrient availability and therefore growth.
Clearly this hypothesis requires further investigation. To
our knowledge, there is no information on plant tissue
culture and colloidal particles in the literature.
The ash composition and quality of the cow dung
could be a factor. The makeup of dung depends on the
diet of the cow and the location, and perhaps, even its
health condition, so that specific proportions of minerals
remaining in the ash could vary by batch and
consequently influence plant growth differently each
time. Changes in the mineral composition of the medium
alone can direct or alter growth without the necessity of
growth regulators (Ramage and Williams 2002; Niedz
and Evens 2007). Whether or not this was the case in the
present study should be verified.
Finally, evaluating the suitability of agnihotra ash/
cow dung ash in more economically important crops
such as banana, coconut, and abaca, in which
propagation relies almost exclusively on tissue culture
technology, can be a worthwhile undertaking.
ACKNOWLEDGEMENT
The authors wish to thank the DOST-ASTHRDP-NSC
Scholarship for the funds used in this research and the
Department of Agriculture-Southern Tagalog Integrated
Agricultural Research Center (STIARC) in Lipa City,
Batangas, Philippines for the adlay seeds.
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