Effect of Benzylaminopurine (BAP) on in vitro Proliferation and Growth of Pineapple (Ananas comosus L. Merr.) cv. Smooth Cayenne
Abdelhamid M. Hamad
The effect of 6-benzylaminopurine
(BAP) at concentration range of 0 to 3.75 mg L-1 applied at
concentration difference of a fixed increment of 0.25 mg L-1
on the in vitro shoot formation and average and total of shoot
length and weight per explant of smooth cayenne pineapple were evaluated.
BAP significantly affected the average of shoot number, length and weight
but did not affect the total length and total weight per explant. The
maximum shoot formation (12 shoots/explant) occurred at 4 concentrations
of BAP (1.75, 2.0, 2.25 and 3.5 mg L-1) while tallest (25 mm)
and heaviest (0.60 g) shoots obtained on MS hormone free medium. Shoot
formation pattern showed two sudden increases in shoot formation. The
first occurred at 1.25 and second at 3.50 mg L-1 and two sharp
declines at 2.5 and 3.75 mg L-1. Identifying of the concentrations
with contrasting effect where an increase and decline in the shoot formation
pattern occur provide an essential base from which an informative successful
histological and biochemical studies could be designed to elucidate shoot
In vitro multiplication of pineapple shoots could be achieved
in medium enriched with BAP alone (Be and Debergh, 2006), combination
of two hormones, BAP plus, naphthalene acetic acid (NAA) (Firoozabady
and Gutterson, 2003), indole acetic acid (IAA) (Hamad and Taha, 2008),
indole butyric acid (IBA) (Boxus et al., 1991) and 2,4-dichlorophenoxy
acetic acid (2,4-D) (Liu et al., 1989) and combination of three
hormones, BAP plus two auxins as NAA and IBA (Soneji et al., 2002),
NAA and IAA (Mathews and Rangan, 1979) and IAA and IBA (Teixeira et
al., 2006). Single application of BAP is simpler and cheaper and should
be favored over combination of two and three hormones. However, the optimum
concentration of BAP was not agreed upon. BAP at 1.0 (Be and Debergh,
2006), 1.5 (Almeida et al., 2002), 2.0 (Bhatia and Ashwath, 2002),
2.5 (Smith et al., 2002), 3.0 (Firoozabady and Gutterson, 2003)
and 4.0 mg L-1 (Omokoio et al., 2001) were recommended
for multiplication of pineapple. It seemed that the optimal BAP concentration
lay between 1.0 and 4.0 mg L-1. However, in all of these studies
either the tested concentrations were as low as 2 concentrations (Almeida
et al., 2002) or the difference among the tested concentrations
range (the increment) was too large, 2.0 mg L-1 (Omokoio et
al., 2001) or both to draw a conclusive conclusion. While BAP concentration
up to 1.0 mg L-1 was thoroughly investigated at a concentration
differences of 0.2 (Be and Debergh, 2006) and 0.25 mg L-1,
(Aydieh et al., 2000) a concentration difference of 0.5 mg L-1
used for testing concentrations range up to 1.5 (Zepeda and Sagawa, 1981),
2.0 (Fernando, 1986), 2.5 mg L-1 (Bhatia and Ashwath, 2002).
Concentration up to 3 mg L-1 tested at concentration difference
of 1.5 mg L-1 (Almeida et al., 2002) and up to 12 mg
L-1 at concentration difference of 2.0 mg L-1 (Omokoio
et al., 2001).
Being about 30 different hormone treatments have been reported for multiplication
of pineapple, raise the question of which should be used. Interestingly,
although in most of these treatments, whether the hormones applied singly
or in combinations, the difference was concentration more than the types
of hormones, the effect of BAP concentrations above 1.0 mg L-1
on pineapple cultures were roughly investigated (few levels and large
increment). It is well known that the same hormone could promote at one
concentration and inhibit at other and a minute difference could draw
the line between the two effects. Hence, it is highly likely that the
best concentration was left out and not being included in these studies
of pineapple. In fact this case was proven to be true for caster bean.
Using wider concentration range enabled Sujatha and Reddy (1998) to select
treatment that increased castor proliferation rate five times higher than
previously reported. In addition, due to using of few concentrations and
either unequal or too large increments, the shoot formation pattern in
response to different BAP in all of the previous studies of pineapple
was neither discussed nor it could be visualized from the reported data.
We think that this area needs to be covered not only for optimization
of multiplication but most importantly for pin point of the concentrations
which would have contradicting effect on the shoot formation pattern.
From there further studies could be conducted to elucidate the mode of
hormone effect and mechanism of shoot formation.
The objectives of this study were first to test the effect of singly
applied BAP at a wider range of 0 to 3.75 mg L-1 and smaller
increment of 0.25 mg L-1 than those reported before on the
shoot formation per explant. Secondly, to evaluate the amount of growth
in term of average length and weight of the produced shoots and total
length and weight that could be obtained from single explant for assessment
of biomass production. Thirdly, to follow the shoot formation pattern
and determine the concentrations at which the BAP promote and inhibit
the proliferation and growth of pineapple and how much change would occur
in response to one or several increase of a fixed increment of 0.25 mg
L-1 of BAP.
MATERIALS AND METHODS
Three liters of MS medium (Murashige and Skoog, 1962) were prepared from
stock solutions. The medium divided into 16 beakers (180 mL each). No
hormone added to the first beaker and BAP at 0.25, 0.50, 0.75, 1.00, 1.25,
1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50 and 3.75 mg L-1
were added to beakers No. 2-16, respectively. The medium pH adjusted to
5.7 and the content of each beaker dispensed equally (20 mL) into 9 glass
jars (5x15 cm). Agar at 7 g L-1 was added to each jar, the
jar closed with autoclavable lid and the medium autoclaved at 121 °C and kg cm-2
for 25 min. Stock cultures that were maintained by subculturing every
75 days on agar solidified MS medium enriched with BAP at 2.3 mg L-1
were used as explants source for this study. One shoot was cultured per
each jar under aseptic conditions. The cultures incubated under a constant
temperature of 25 °C ± 2 and 16 h of artificial light provided
by fluorescent lamp. After 60 days, the cultures were taken of the incubation
room and the multiple shoots were picked up, weighted and separated into
individual shoots for counting number and measuring the shoot length.
The weight of multiple shoots per each culture considered as total weight
per explant. The length of shoots summed and considered as total length
per explant. Average weight and length of shoots computed from dividing
total weight and total length by number of shoots per explant. Each treatment
consisted of nine cultures (3 cultures per replicate) and the data recorded
from each three of the nine cultures summed and divided by 3 to obtain
the average value for each replicate. Analysis of variance, treatments
means separation by Duncan Multiple Range Test, correlation and regression
analysis was done at p<0.5 using SPSS 11.0 statistical package.
Table 1 shows that the shoots formation increased in
response to every 0.25 mg L-1 increase in concentration of
BAP up to 1.0 mg L-1 at almost steady rate (1 shoot/each 0.25
mg L-1 increase in concentration). Then an increase of 0.25
mg L-1 resulted in a sudden surge in shoot formation and the
shoot number rose from 6 to 10 shoots/explant as the concentration rose
from at 1.0 to 1.25 mg L-1. After that, the rate of shoot formation
remained almost stable around 11 shoots per explant for four consecutive
increases in the concentration of BAP by 0.25 mg L-1 till the
concentration reached 2.25 mg L-1. Then an increase of 0.25
mg L-1 caused a sharp decline in shoot formation and the shoots
number decreased from 12 to 8 shoots/explant in response to increase of
BAP concentration from 2.25 to 2.5 mg L-1.
Effect of BAP concentrations on shoots No., average
shoot length (Lx), shoot weight (Wx) and total shoots length (LT)
shoots weight (WT) per explant of smooth cayenne pineapple
Number represent means of 9 shoot buds individually
cultured in 20 mL of agar solidified (7 g L-1) MS medium
containing 30 g L-1 of sucrose and enriched with BAP at
different concentration (mg L-1) for 60 days. pH adjusted
to 5.7. Means of the same column followed by same letter(s) were not
significantly different at p = 0.05 using Duncan`s multiple range
test. ns (No significant effect on total weight (p = 0.2496) and total
length (p = 0.2135)
and regression equations among
BAP at concentration range of 0.0-3.75 mg L-1
and the growth
parameters of smooth cayenne pineapple cultured on agar solidified
full strength MS medium for 60 days
Lx: Average length of shoot, LT: Total length of shoots
per explant, Wx: Average fresh weight of shoot, WT: Total fresh weight
of shoots per explant
The rate again remained
stable at 8 shoots per explant for four consecutive increases in BAP concentration
by 0.25 mg L-1 till the concentration reached 3.25 mg L-1.
After that increase of concentration by 0.25 mg L-1 from 3.25
to 3.5 mg L-1 reversed the shoot pattern and a second surge
of shoot formation from 7 to 12 shoots per explant occurred. However,
the potential for shoot formation the explants had just gained was lost
upon raising the concentration to 3.75 mg L-1. Generally, there
were two sharp increases and decreases in shoot formation. The first increase
occurred as the concentration raised from 1.0 to 1.25 mg L-1 BAP and the second increase as the concentration raised from 3.25 to 3.5
mg L-1 while the first decrease occurred as the concentration
raised from 2.25 to 2.5 and the second decrease when the concentration
raised from 3.5 to 3.75 mg L-1. There were no significant difference
between shoot number per explant at 1.75, 2.0, 2.25 and 3.5 mg L-1 of BAP and all induced the highest shoot formation (12 shoots/explant)
and no significant differences between shoot number per explant at 2.5,
2.75, 3.0 and 3.75 mg L-1 of BAP and all resulted in intermediate
shoot formation (8 shoots per explant). At any concentration below 1.0
mg L-1, the shoot formation was less than 6 shoots per explants
and each increase of 0.25 mg L-1 resulted in significant increase
in number of shoots per explant. Regression analysis (R2 =
0.71) indicated that 71% of the difference in shoots formation was caused
by differences in the concentration of BAP and the relation between BAP
concentration and shoot formation was cubic polynomial relationship (Table
2). Correlation analysis indicated that the relation between shoot
formation and BAP concentrations was positive (r = 0.63) and the shoot
increased as the BAP concentration increased but was negative with average
length (r = -0.83) and weight (r = -0.76).
The tallest (25 mm) and heaviest (0.67 g) shoots obtained in hormone
free and at 0.25 mg L-1 and the shortest (7 mm) and lightest
(0.13 g) shoots at 1.25 mg L-1. The shoot length decreased
as the BAP concentration increased up to 0.5 mg L-1 while shoot
weight decreased as concentration increased up to 1.25 mg L-1.
Concentrations above 0.5 and 1.25 up to mg L-1 did not cause
any significant difference in average shoot length and shoot weight/explant
respectively (Table 1). Regression coefficient of shoot
length (R2 = 0.93) and shoot weight (R2 = 0.87)
indicated that 93% of differences in shoot average length and 87% of the
differences on average shoot weight were due to BAP concentrations (Table
2). Correlation analysis between BAP concentrations and both of average
shoot length (r = -0.73) and average shoot weight (r = -0.54) indicated
a negative relationship. Both of the shoot length and weight average correlated
positively with each other (r = 0.82) and negatively (r = -83 and -76,
respectively) with shoot formation. The effect of different BAP concentrations
on average shoot length and average weight per explant seemed to follow
a similar pattern. Both responded negatively and only to low concentration
range (0.25 to 1.25 mg L-1) of BAP.
BAP concentrations, on the other hand, had no significant effect on total
length (p<0.2135) and total weight (p<0.2496) of shoots per explant.
Nevertheless, the largest total length (109 mm) and heaviest total weight
(2.61 g) obtained on medium enriched with BAP at 3.5 mg L-1 and
the lowest total length (37 mm) and total weight (0.99 g) obtained on
hormone free medium (Table 1). The total length and
total weight of shoots showed similar pattern but opposite to the pattern
of the average shoot length and weight per explant. Both of the total
length and weight were not affected by BAP concentrations, correlated
positively with shoot formation (r = 93 and 80) and with each other (r
= 85) and negatively with average shoot length (r = -66 and -58) and with
average shoot weight (r = -66 and 45).
The optimal concentration of BAP for multiplication of pineapple (Ananas
comosus L. Merr.) cv. smooth cayenne in solid MS appeared to be 1.75,
2.0, 2.25 and 3.5 mg L-1. Each resulted in the highest number
of shoot formation (12 shoots/explant) and equal shoot length of 8 mm
long and about 0.18 g/explant (average weight/explant) and over 1.5 g
per culture (total weight/explant) per 60 days of incubation.
The main goal of tissue culturist is to optimize the multiplication.
However, in most of the time they follow try and error approach to select
best concentration of a one hormone or best hormone type applied singly
or in combinations. Sujatha and Reddy (1998) used this approach to optimize
the multiplication of castor bean and come up with hormone treatment that
resulted in multiplication five times higher than previously reported
treatments. The 12 shoots per explant of pineapple obtained in this study
in agar solidified MS enriched with BAP at 1.75, 2.25 and 3.5 mg L-1
is higher than the previously reported 10 (Sripaoraya et al., 2003)
and 7 shoots per explant (Bhatia and Ashwath, 2002) obtained in response
to BAP at 2.0 mg L-1 and the 9 (Be and Debergh, 2006), 7 (Aydieh
et al., 2000) and 3 shoots per explant (Zepeda and Sagawa, 1981)
obtained in response to BAP at 1.0 mg L-1 and the 10 shoots
(Firoozabady and Gutterson, 2003) in response to 3.0 mg L-1.
The result did not support the use of BAP at 1.5 mg L-1 suggested
by Ameida et al. (2002) and 2.5 mg L-1 suggested by
Smith et al. (2002).
Although trial and error approach is very important and the most common
used approach, the most optimal treatment would not be achieved unless
the mode of hormone action and shoot formation mechanism is elucidated.
Understanding of mode of action and mechanism require histological and
chemical analysis of the endogenous content of the explants in response
to different concentrations and type of hormone. The success on this approach
depends mainly on proper selection of the concentration of the added hormone
and time at which these analyses should be taken. Hence, the first essential
step is to determine the growth and shoot formation pattern. Surprisingly
in most of pineapple studies, shoot formation pattern in response to a
continuous increase in concentrations by one fixed unit of concentration
over wide range were either totally ignored or it could not be discern
from the limited reported data. Exogenously applied hormone stimulates
synthesis of endogenous hormones in pineapple leaf culture and histological
and chemical studies confirmed strong association between sharp increase
in the endogenous IAA (indole acetic acid) and 2iP (isopentenyladenine)
content and formation of shoots (Hamasaki et al., 2005; Mecier
et al., 2003). In these studies, a fixed and arbitrary selected
hormone concentration was used. If two different concentrations one inhibit
and other promote shoot formation were used the results would be more
informative. In this study we think that the first step has been taken,
the changes in shoot formation were followed at fixed unit of concentration
increase (0.25 mg L-1) and the different concentrations at
which the BAP promote and inhibit the shoot formation have been identified.
It is very interesting to note that a 0.25 mg L-1 difference in
concentration of BAP could reverse the mode on either positive or negative direction
and the effect seemed to follow symmetrical trend. Raising the concentration
by 0.25 mg L-1 from 1.0 to 1.25 and from 3.25 to 3.5 resulted in
sudden sharp increase in the shoot formation capacity by 4 more shoots (30%
more) and the capacity just gained maintained for 4 consecutive 0.25 mg L-1
increases in concentration to be lost by the fifth. Raising the concentration
by the same amount (0.25 mg L-1) from 2.25 to 2.5 and from 3.5 to
3.75 mg L-1 caused on the contrary drastic loss of 4 shoots and the
loss on the capacity persisted for 4 consecutive increases in the concentration
to be reversed by the fifth. It appeared that 4 consecutive increases in concentration
by 0.25 mg L-1 are required before the fifth could cause sudden sharp
increase or drastic decrease in shoot formation. Nielsen et al. (1995)
proposed one receptor and two binding sites model for cytokinin action in plant
cell. Functioning receptor-hormone complex is the one in which the sites occupied
by the endogenous hormones. High concentrations of exogenous hormone compete
with endogenous hormone for the active sites or change the conformation of the
receptor and reduce the total number of functioning complex. The ratio between
the exogenous and endogenous hormone and the natural turn over of functional
complex determine the shoot formation pattern. The shoot formation pattern could
also relate to changes in the other chemical content within the explants and
the different concentrations of the hormone may affect the uptake of nutrient
and medium pH differently. Chemical analysis of agar-solidified medium was successfully
used for identification of anti-hyperhydricity factors and facilitates commercial
micropropagation of radiata pine (Nairn et al., 1995). Similar approach
could be proven successful for pineapple. Comparing of histological and chemical
analysis of explants and MS components in medium enriched with BAP at 1.25 and
3.5 with that from MS enriched with BAP at 2.5 and 3.75 would probably shed
some light on the physiology of in vitro shoot formation and growth of
The pineapple tissue culture is usually done for propagation purpose
and the best treatment judged by the rate of shoot formation. Other parameters
such as total or average fresh weight are rarely reported. As the goal
was propagules production, neglecting of weight are understandable. However,
since there is possibility for extraction of bromelain for medical purpose
and use of biomass for animal feeding reporting of weight would be more
important than any other parameters. The heaviest shoot (average weight
per shoot) obtained in medium contained BAP at 0.25 mg L-1
but there were no significant different in the total weight per explant
(weight/culture). Hence, for biomass production hormone addition is not
required. On the contrary, Pereze et al. (2003) obtained higher
fresh weight, protein content and protease activity on response to BAP
at 0.5 mg L-1. Be and Debergh (2006) reported that although
BAP at 1.0 mg L-1 and combination of BAP at 1.0 plus IBA at
0.5 mg L-1 induced equal number of shoots, the second treatment
doubled the total fresh weight/culture. Existing of positive correlation
between shoot numbers, total weight, total length (Table
2) indicated that a system for optimum production of both propagules
and biomass could be developed using MS medium enriched with proper concentration
of BAP. In addition, in cases when high explants density are used and
in large scale production counting of individual shoot is not practical.
The high correlation between number and total weight of shoots (r = 0.80)
indicated that rather than individual counting, the shoot number could
possibly estimation as function of total weight according to the model
presented (Table 2). Firoozabady and Gutterson (2003)
counted the number of shoots in specific unit of weight and used that
ratio for estimation of shoot number from a total weight obtained in bioreactor
system. Salehi and Khosh-Khui (1997) suggested a model by which the shoot
length after one week in culture could be used for estimation of the number
of shoots that miniature rose would produce after four weeks of incubation.
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