International Journal of Biotechnology and Bioengineering Research.
ISSN 2231-1238, Volume 4, Number 5 (2013), pp. 465-472
© Research India Publications
http://www.ripublication.com/ ijbbr.htm
Interaction between GA and Ethrel in Inducing Female
Flowers in Jatropha Curcas
Vijay Makwana1 and Pushpa Robin1*
1
Department of Biochemistry, The Maharaja Sayajirao University of Baroda,
Vadodara, Gujarat, INDIA.
Abstract
Jatropha curcas is a plant with tremendous economic potential as a
source of biodiesel. It is a monoecious plant with staminate and
pistillate flowers on same inflorescence. The number of staminate
flowers is higher than pistillate which results in low fruit yield.
Phytohormones such as Gibberellin and Ethylene play an important
role in modulating flower development. Gibberellin and Ethylene play
different role in sex alteration in different plant species. There exists a
crosstalk between Gibberellin, Ethylene and Auxin during growth and
development. In this study, we report the exogenous application of GA
and Ethrel and its impact on femaleness. A correlation between
endogenous level of Auxin, Gibberellin to flowering and fruit yield in
Jatropha curcas has been attempted. There was considerable decrease
in flowering and fruit yield after the application of Ethrel (15 ppm)
while GA 100 ppm significantly increases flowering and fruit yield
compared to other treatments. The changes seen in endogenous level of
Auxin and GA in response to GA and Ethrel application in various
stages of inflorescence and its effect on number of female flowersis
discussed.
Keywords: GA (Gibberellin), Ethrel; Flowering; Fruit yield; Jatropha
curcas.
1. Introduction
Jatropha curcas is a plant with tremendous potential as a source of biodiesel. It is a
monoecious shrub having staminate (male) and pistillate (female) flowers on same
466
Vijay Makwana & Pushpa Robin
inflorescence. A typical inflorescence has more male than female flowers, average
male to female flower ratio being 29:1 (Solomon and Ezradaman, 2002). Increasing
female flowers could result into greater seed yield and tremendous yield of oil. A large
number of factors are responsible for sex expression and sex alteration in plants
(Dellaporta et al, 1994; Stehlik et al, 2008). The biochemical mechanism for sex
expression is largely unclear. Among various factors, phytohormones play a very
important role (Durand and Durand, 1991). Of various phytohormones, Auxin,
Ethylene and Gibbrellin (GA) play an important role in regulation of flower sex
expression (Tanimoto 2007). Effect of exogenous phytohormones on floral sex ratio
depends on species of plant. GA promotes female flowers in maize and castor bean; on
the contrary, it increases masculine features in hemp, spinach and cucumber (Pharis
and King, 1985). Ethylene increases femaleness in cucurbits species such as cucumber
and watermelon. In some plant species such as Arabidopsis and Tomato, GA
deficiency leads to male sterility because of abnormal anther development (Goto and
Pharis, 1999). Ethylene affects GA biosynthesis and its interaction with GA governs
the stability of DELLA proteins and hence flowering. It is reported that Ethylene via
its effect on GA biosynthesis results in repression of two flowering genes, LEAFY and
SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (Grauwe et al, 2008).
Our earlier work has shown the effect of GA and 2, 4-D on influencing flowering
in Jatropha curcas (Makwana et al, 2010a, 2010b). In that study at higher
concentration of GA we reported withering of flowers and fruits. Ethylene has been
shown to be the culprit in causing withering of flowers and abscission of fruits. To
elucidate if GA effects involve ethylene in this study, we have seen the effect of GA
and Ethrel on endogenous levels of the phytohormones and correlated it with changes
in flowering and flower sex ratio.
2. Materials and Methods
2.1 Experimental plants
Jatropha curcas plants (14 month old) of Padra region in Vadodara, India, were
selected for experiments. Solutions of GA (100 and 1000 ppm) and Ethrel (15 and 25
ppm) were prepared by dissolving GA and Ethrel in small volume of isopropyl alcohol
and volume made up with demineralized water after adjusting pH to 7.5–7.8. A few
drops of Tween-20 as surfactant were added to the solution. Plants sprayed with
demineralized water containing surfactant were considered as control. The time of
spray was early morning and spraying was initiated from the time foliar bud emerged.
Each inflorescence received three sprays of equal volume of solution at an interval of 5
days. Four plants were taken per treatment. Total number of flowers and sex ratio were
calculated 1 week after the last spray whereas fruit yield was measured 1 month after
the last spray. Test and control plants were tagged with appropriate labels to follow
flower development. Total fruit yield was calculated by taking observations for 3
months at an interval of 1 month.
Interaction between GA and Ethrel in Inducing Female Flowers in Jatropha
467
2.2 Determination of GA and Auxin
GA levels were determined by Holbrook et al, 1961. Tissue of specific stage of
inflorescence (0.5 gm) was ground to a homogenized paste in a mortar with pestle,
along with 10 ml Phosphate buffer. This mixture was then centrifuged at 5000 g for 20
min at 4oC. The supernatant was recovered and its pH was adjusted to 1 by 1N HCl.
GA was extracted from supernatant by ethyl acetate. 3.75 N HCl was added to the
extracted GA and absorbance at 254 nm was recorded at 20 s intervals for 2 min.
Temperature was kept at 20 ± 0.5 ◦C during the process. Auxin was determined by
Gordon and Weber, 1951. Tissue (0.5 gm) was ground in a mortar with pestle, together
with 0.01M EDTA and 0.02M Sodium diethyldithiocarbamide in 80% ethanol. This
was transferred to test tube, kept in ice bath for 15-20 min, shaking intermittently. This
mixture was centrifuge at 5000 g for 20 min at 4oC. Auxin was estimated in the
Supernatant at 530 nm. All the values are reported as mean ± SEM. Statistical analysis
of data was done using student’s t-test.
3. Results and Discussion
Both GA and Ethrel treatments altered flowering pattern. GA (100, 1000 ppm) and
Ethrel 25 ppm, resulted in an significant increase in total flowers, female:male flower
ratio and female flowers (Table 1). The total number of flowers increased by 2 and 3
fold on 100 ppm and 1000 ppm GA treatment respectively while Ethylene 25 ppm
increased it by 0.2 fold. This increase was mostly due to increase in number of female
flowers which increased by almost four to five folds on GA 100 and 1000 ppm while it
was 1.5 fold increase by Ethrel 25 ppm. This alteration resulted in an appreciable and
desirable increase in ratio of female:male flowers. Ethrel 15 ppm showed significant
decrease in total flowers and female: male flower ratio. Role of GA in regulation of
flowering is well studied and the molecular events involved have been deciphered. GA
regulates development of flowers by activation of LFY and AP1 genes (Jack, 2004).
Apart from increasing, total number of flowers and female:male ratio, GA and Ethrel
also alters the process of flower development. Application of Ethrel 15 ppm, delayed
duration of flower development from 36 to 78 days, on the other hand GA 1000 ppm
decreased time duration from 36 to 22 days (Table 2).
Table 1: Effect of Exogenous GA and Ethrel on Female:
Male Flower Ratio in Jatropha curcas.
Treatment Numbe Number of Total flowers Total
Total
Female:
(ppm)
r of
Inflorescenc
in
Female Male
Male
Plants es observed Inflorescences flowers flowers flower ratio
Control
4
10
1395 ± 0.378 51 ±
1344 ± 1:26
0.481
8.432
468
Vijay Makwana & Pushpa Robin
Ethrel
15 ppm
4
10
988 ±
0.326***
Ethrel
25 ppm
4
10
1495 ±
0.237***
GA 100
ppm
4
10
3117.1 ±
0.245***
GA 1000
ppm
4
10
3341.9 ±
0.242***
37 ±
0.238**
*
70 ±
0.247**
*
195 ±
0.263**
*
245 ±
0.283**
*
951 ±
7.246**
*
1425 ±
10.34**
*
2923 ±
9.825**
*
3097 ±
16.95**
*
1:25
1:20
1:15
1:13
Values are mean ± SE, *** indicates significantly different at P<0.001 as compared to the
corresponding control.
The stages of flower development were characterized as shown in Fig. 1. There
was a delay in the initial four stages of flower development by Ethrel treatment as
compared to control. Femaleness could be as a result of levels of phytohormones. The
levels of GA and auxins were monitored in these plants. Amongst other biochemical
changes brought about by GA and Ethrel application was a pronounced change in
endogenous level of Auxin and GA during the initial stages of inflorescence
development (Stage 1 and 2) (Fig. 2 and 3).
Stage-I
Stage-II
Figure 1: Initial stages of Inflorescence development were observed and duration for
flower development calculated from the emergence of floral bud. Stage-I is initial
appearance of floral bud and Stage-II is distinct appearance of floral bud.
Interaction between GA and Ethrel in Inducing Female Flowers in Jatropha
**
*
***
*
**
469
***
***
**
*
*
Figure 2: Effect of GA and Ethrel on endogenous level of GA in Jatropha curcas.
Values are mean ± SE; *, **, *** indicates significantly different at P<0.05, P<0.01
and P<0.001 as compared to the corresponding control. (n = 3)
Table 2: Effect of GA and Ethrel on time scale of inflorescence development in
Jatropha curcas Emergence of floral bud was considered as day zero, Values are
mean ± SE, **, *** indicates significantly different at p<0.01 and p<0.001 as
compared to the corresponding control.
Treatment
Control
Ethrel
15 ppm
Ethrel
25 ppm
Duration (in days) for inflorescence development
Initial
Distinct
Distinct Opening Opening Complete
appearance appearance Male and of Male
of
bloom
of floral
of flower
Female flowers Female
bud
bud (Stage flower
(Stage
flowers
(Stage I)
II)
bud
IV)
(Stage V)
(Stage
III)
9.00 ± 0.577 6.333 ±
4.667 ±
6.160 ± 9.467 ± 36.20 ±
0.333
0.333
0.333
0.333
1.068
24.00 ±
26.33 ±
14.20 ±
8.47 ±
5.00 ±
78.01±
0.675 **** 0.4333*** 0.365*** 0.361*** 1.447*** 1.198***
22.00 ±
17.33 ±
15.60 ±
7.10 ±
4.90 ±
66.93±
0.5774 *** 0.333***
0.467*** 0.583*** 1.365*** 1.171***
470
GA
100 ppm
GA1000
ppm
Vijay Makwana & Pushpa Robin
5.00 ±
0.447***
4.20 ±
0.374***
5.80 ±
0.365**
3.60 ±
0.374***
4.40 ±
0.509**
4.20 ±
0.583**
5.10 ±
0.871***
5.60 ±
1.020***
5.60 ±
1.02***
5.20 ±
1.249***
25.90 ±
1.02***
22.80 ±
1.249***
In GA 100 ppm treatment, there was an increase in the endogenous level of Auxin
and GA in stage-1 than other treatments. This could be correlated to increased
flowering and fruit yield. Increased Auxin promotes GA responses via destabilization
of DELLA. Ethrel treatment shows a decreased level of Auxin in initial stage of
inflorescence. Ethrel 25, ppm shows increased flowering and fruit yield than Ethrel 15
ppm due to decreased level of endogenous Auxin in initial appearance of floral bud. It
suggests that increased Ethrel treatment decreases Auxin level which upregultes LFY
and AP1 genes that are MADS box transcription regulator, hence flowering is
increased.
a
***
b
***
b
***
a
***
*
***
*** ***
Figure 3: Effect of GA and Ethrel on endogenous level of IAA in Jatropha curcas.
Values are mean ± SE; *, ***P<0.05 and P<0.001 as compared to the corresponding
control. ‘a’ and ‘b’ shows significant different at P<0.01. (n=3).
Interaction between GA and Ethrel in Inducing Female Flowers in Jatropha
471
***
*
*
***
Fig. 4: Effect of GA and Ethrel on Fruit yield in Jatropha curcas. Numbers of fruits
calculated 1 month after last spray. Values represented is mean of 4 replicates, bars
indicate SE, *, ***P<0.05 and P<0.001, respectively (n=4).
Phytohormone treatment not only increases flowering but also effects fruit yield
(Fig. 4). GA 100 ppm treatment resulted in increased fruit yield. However, the
increased flowering in 1000 ppm GA and Ethrel 25 ppm did not translate into
increased fruit yield because of senescence and programmed cell death. One of the
important initiators of senescence is endogenous release of hydrogen peroxide
(Strother 1988; Makwana et al, 2010a).
4. Conclusion
In summary, GA treatment of Jatropha curcas foliar bud increases the number of
female flowers and hastens flower development due to increased endogenous level of
GA and Auxin. However, lower dose of Ethrel treatment decreases the number of
flowers and delays the flower development due to decreased endogenous level of
Auxin than other treatment. Increased flowering and fruit yield by Ethrel treatment is
dose dependent. Higher number of female flowers at 1000 ppm of GA did not translate
into high fruit yield due to withering of immature fruits and flowers. Withering of
fruits and flowers could be due to cell death mediated by hydrogen peroxide release at
the zone of abscission.
472
Vijay Makwana & Pushpa Robin
5. Acknowledgment
The authors are thankful to Dr. Bhaskar Vyas, Vadodara for allowing use of their
Jatropha curcas plantation for this study. PR also acknowledges financial support
from UGC, New Delhi, India for this work.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
S L Dellaporta, A Calderon-Urrea (1994), The Sex Determination Process in
Maize, Science, 266, pp 1501-1505.
B Durand, R Durand (1991), Male sterility and restored fertility in annual
mercuries, relations with sex differentiation, Plant Science, 80, pp107-118.
S A Gordon, R P Weber (1951), Colorimetric estimation of indole-acetic acid,
Plant Physiol, pp 26:192–195.
N Goto, R P Pharis (1999), Role of gibberellins in the development of floral
organs of gibberellin-deficient mutant, ga1-1, of Arabidopsis thaliana, Can J
Bot, pp77:944–954.
L D Grauwe, J Dugardeyn, D V D Straeten (2008), Novel mechanisms of
ethylene-gibberellin crosstalk revealed by the gai eto2-1 double mutant.,Plant
Sign Beh, 3, pp 1113-1115
A Holbrook, W Edge, F Bailey (1961), Spectophotometric method for
determination of gibberellic acid, Adv Chem Ser, 28, pp 159-167.
T Jack (2004), Molecular and Genetic Mechanisms of Floral Control, Plant
Cell, 16,pp S1-S17.
V Makwana, P Shukla, P Robin (2010a), GA application induces alteration in
sex ratio and cell death in Jatropha curca,. Plant Growth Regul , 61, pp121–
125.
V Makwana, P Shukla, P Robin (2010b), Comparing potential of GA and 2, 4D in increasing fruit yield from Jatropha curcas, J Biofuels, 1, pp1157-162.
R P Pharis, R W King (1985), Gibberellins and reproductive development in
seed plants, Ann Rev Plant Physiol, 36, pp517–568.
R Solomon, V Ezradanam (2002), Pollination ecology and fruiting behaviour
in a monoecious species, Jatropha curcas L. (Euphorbiaceae), Current
Science, 83, pp1395-1398.
I Stehlik, J Friedman, S C H Barrett (2008), Environmental influence on
primary sex ratio in a dioecious plant, Pro Nat Ac Sc, 105, pp10847-10852.
S Strother (1988), The Role of Free Radicals in Leaf Senescence,
Gerentology, 34, pp151-156
T Tanimoto (2007), Modification of Sex Expression in Sagittaria latifolia by
the Application of Gibberellic Acid and Paclobutrazol,. J Japan Soc Hort Sci,
76, pp47-53.