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*

1Department 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

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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

(ppm) Numbe

r of Plants

Number of Inflorescences observed

Total flowers in

Inflorescences

Total Female flowers

Total Male

flowers

Female: Male

flower ratio Control 4 10 1395 ± 0.378 51 ±

0.481 1344 ± 8.432

1:26

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Ethrel 15 ppm

4 10 988 ± 0.326***

37 ± 0.238***

951 ± 7.246***

1:25

Ethrel 25 ppm

4 10 1495 ± 0.237***

70 ± 0.247***

1425 ± 10.34***

1:20

GA 100 ppm

4 10 3117.1 ± 0.245***

195 ± 0.263***

2923 ± 9.825***

1:15

GA 1000 ppm

4 10 3341.9 ± 0.242***

245 ± 0.283***

3097 ± 16.95***

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

Duration (in days) for inflorescence development Initial

appearance of floral

bud (Stage I)

Distinct appearance

of flower bud (Stage

II)

Distinct Male and Female flower

bud (Stage

III)

Opening of Male flowers (Stage

IV)

Opening of

Female flowers

(Stage V)

Complete bloom

Control 9.00 ± 0.577 6.333 ± 0.333

4.667 ± 0.333

6.160 ± 0.333

9.467 ± 0.333

36.20 ± 1.068

Ethrel 15 ppm

24.00 ± 0.675 ****

26.33 ± 0.4333***

14.20 ± 0.365***

8.47 ± 0.361***

5.00 ± 1.447***

78.01± 1.198***

Ethrel 25 ppm

22.00 ± 0.5774 ***

17.33 ± 0.333***

15.60 ± 0.467***

7.10 ± 0.583***

4.90 ± 1.365***

66.93± 1.171***

** **

**

* *

***

*** **

***

Vijay Makwana & Pushpa Robin

470

GA 100 ppm

5.00 ± 0.447***

5.80 ± 0.365**

4.40 ± 0.509**

5.10 ± 0.871***

5.60 ± 1.02***

25.90 ± 1.02***

GA1000 ppm

4.20 ± 0.374***

3.60 ± 0.374***

4.20 ± 0.583**

5.60 ± 1.020***

5.20 ± 1.249***

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.

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).

***

***

a ***

b ***

*** *** ***

* a

b

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.

***

*

***

*

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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

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