Jatropha curcas is a potential bio-fuel plant with highly variable fruit and seed yield. Female flowers are far lesser in number as compared to male flowers which is the main reason behind the low fruit yield. Effect of four different Plant Bio-Regulators (PBRs) viz. 6-Benzyladenine (BA), Inole-3-butyric Acid (IBA), Indole-3-Acetic Acid (IAA) and Gibberelic acid (GA₃) were studied on the male: female flower ratio, fruit yield and seed yield of Jatropha curcas at DIBER Project site, Mhow (MP), India. The PBR’s concentration range was kept from 50 ppm to 250ppm.This experiment was conducted during 2013, 2014 and repeated in 2015 to confirm the results. Exogenous application of Plant Bio-Regulators had a positive effect on the number of female flowers, total number of flowers and number of fruits per plant. BA (100ppm) application modifies the male: female flower ratio significantly from 13:1 to 5:1. Consequently, 3 to 3.5 fold increases in fruit numbers and a 3.1 fold increase in final seed yield were observed. IAA (100ppm), IBA (100ppm) and GA₃ (50ppm) were also found enhancing the fruit and seed yield as compared to control plants. The study revealed that 3 to 3.5 times seed yield of Jatropha curcas can be increased by exogenous application of PBRs, which is a significant finding to improve the availability of feed stock for biodiesel production from the Jatropha plantation.

Keywords: Jatropha curcas, plant bio-regulators, BA, GA₃,IBA, IAA

PBRs, plant bio-regulators; BA, 6-benzyladenine; IBA, inole-3-butyric acid; IAA, indole-3, acetic acid; GA₃, gibberelic acid; PPM, part per million; mg, mili gram; kg, kilogram

Jatropha curcas is a drought tolerant, oil seed bearing shrub of family Euphorbiaceae, widely distributed in Central and South America, Africa, India and south East Asia.^1,2 It is found growing in tropics and subtropics.^3–5 Jatropha is a monoecious plant and flowers are unisexual, occasionally hermaphrodite flower also occur.⁵ There is a huge variation in seed yield of this plant (Figure 1). It varies from 0.1 to15 tonnes/ha/year.⁷ Different researchers have reported different amount of oil content ranging from 24% to 60 %.^8–13 Low yield is the main constraint in successful utilization of this oil yielding plant as biodiesel feedstock.

Figure 1 Jatropha fruit bunch.

Figure 2 Prepation for PBR’s spray on field.

Figure 3(A) Effect of Plant Bio- regulators on flower and fruit numbers.

Figure 3(B) Effect of Plant Bio- regulators on flower and fruit numbers.

Studies of exogenous applications of various plant Bio-regulators (PBRs) and analysis of endogenous phyto-hormones showed that PBRs play important roles in floral development.^14,15 Exogenous cytokinin (CK) application has been shown to increase inflorescence meristem activity and promote floral initiation in several species).^16–18 Srinivasan & Mullins^19,20 reported that the tendrils of grape (Vitis vinifera) were converted into inflorescences by application of various CKs. Ohkawa²¹ found that 6-benzyladenine (BA, a synthetic compound with CK activity) treatment had a significant influence on increasing flower numbers of Lilium speciosum, particularly when combined with gibberellins GA4 and GA7. Chen²² showed that flower bud differentiation of Litchi (Litchi chinensis) was significantly promoted by exogenous kinetin application after bud dormancy. The total number of flowers on Jojoba (Simmondsia chinensis) was also significantly increased by treatment with BA.^23,24 Recently, Li & others²⁵ reported that the flower-specific elevation of cytokinin through transgenic expression of an Arabidopsis cytokinin biosynthesis enzyme gene (ATP/ADP isopentenyl transferase 4, AtIPT4) under the control of the APETALA1 (AP1) promoter led to a three-fold increase of flowers in the transgenic plants. PBRs are also important regulators of floral sex determination, which depends on the plant species.^26–28 CKs have been shown to have a feminizing effect on a number of plant species.^26,27 For example, CK induced bisexual (hermaphroditic) flowers of grape (Vitis vinifera)^29,30 and also female flowers of Luffa acutangula³¹ and Luffa cylindrical.³²

To find ways to increase the total number and proportion of female flowers of Jatropha, which may result in increased seed yield.

Yield enhancement studies were carried out at DIBER Bio-fuel Park at Harsola, Mhow (latitude of 22^o 55ʹ N and longitude of 75^o 75ʹE and at an elevation of 556m above mean sea level in District, Indore (Madhya Pradesh) India. Average temperature of the region ranges from 18.1^oC in winter to 32.4^oC in summer and average rain fall of the region is around 304mm. Four different plant Bio-regulators viz. Gibbrellic acid (GA₃)_, Benzyl Adenine (BA), Indole Butyric acid (IBA) and Indole acetic acid (IAA) were evaluated on five year old Jatropha curcas plants at DIBER Bio-fuel Park during April-May 2013-2015 to study their effect on the fruit and seed yield of Jatropha curcas. Exogenous spray of these plant bio-regulators at three growth stages i.e. just before the emergence of flowering buds, during flowering and at the time of fruit setting was done (Figure 2). Each PBR was first dissolved in organic solvent or water whichever is applicable and then stock solutions were prepared in 50ppm, 100ppm, 150ppm, 200ppm and 250ppm concentrations for spray. Solutions were sprayed with knapsack sprayers. Data were recorded for flower and fruit intensity in the PBR’s treated and control plants. Data were collected regarding Male: female flower ratio, number of female flowers per bunch, fruit/flowering branch, fruits/plant, seed/fruit and seed yield per plant and hence per hectare.

Plant Bio-Regulators (PBRs) application

It was observed that plants sprayed with PBR’s showed 2 to 3 fold increase in the flowering intensity as compared to control plants and higher fruit and seed yield was recorded in these plants as compared to control (Figure 3). Plant bio-regulators (GA₃, BA, IBA and IAA) were found to increase the seed yield as compared to the control plants. But application of BA, significantly modifies the M:F ratio from 21:1 to 5:1. Consequently, 3 to 3.5 fold increase in fruit numbers and 3.1 fold increase in final seed yield were observed in the plants sprayed with 100mg/L of BA. This study indicated that the seed yield of Jatropha curcas can be increased by exogenous application of PBRs especially BA (100ppm).

Effect of gibberellic acid (GA₃)

It was also sprayed in 50, 100, 150 and 200ppm concentrations and data regarding male: female ratio, fruits per flowering branch, seeds/fruit and seed yield per plant were recorded. Data revealed that 50ppm GA₃ resulted in highest seed yield per plant as comparison to all other PBRs, though other concentrations also resulted in higher yield per plant as compared to control but 50ppm GA3 significantly enhanced the seed yield per plant (Table 1).

Effect of benzyl adenine (BA)

BA was sprayed in 50,100,150 and 200ppm concentrations and data regarding male: female ratio, fruits per flowering branch, seeds/fruit and seed yield per plant were recorded. Data revealed that 100ppm BA resulted in the highest seed yield per plant though other concentrations also resulted in higher yield per plant as compared to control but 100ppm BA significantly enhanced the seed yield per plant. The results are shown below in the Table 2.

Indole-3- butyric acid (IBA)

Indole-3-butyric acid was also sprayed in 50, 100, 150 and 200ppm concentrations and data regarding male: female ratio, fruits per flowering branch, seeds/fruit and seed yield per plant were recorded. Data revealed that 100ppm IBA resulted in highest seed yield per plant though other concentrations also resulted in higher yield per plant as compared to control but 100ppm IBA significantly enhanced the seed yield per plant (Table 3).

Indole-3-acetic acid

It was also sprayed in 50,100,150 and 200ppm concentrations and data regarding male: female ratio, fruits per flowering branch, seeds/fruit and seed yield per plant were recorded. Data revealed that 100ppm IAA resulted in highest seed yield per plant though other concentrations also resulted in higher yield per plant as compared to control but 100ppm IAA significantly enhanced the seed yield per plant (Table 4).

A comparative performance of all the plant bio-regulators on the fruit and seed yield is depicted below in Figure 4.

Figure 4 Effect of different PBRs on fruit and seed yield (g) per plant.

PBRs mainly act on the newly developing branches and it mainly changes the male: female ratio. It also affects the seed setting in fruits. Generally three seeds are found in each Jatropha fruit but in PGRs applied plants around 5% fruits with 4 seeds in a fruit were also reported and thus enhancing the seed yield (Figure 5).

Figure 5 Change in seed number per fruit due to PBRs spray.

Sex determination in unisexual flowers is a complicated process that is achieved by selectively arresting or aborting pistil or stamen development within a bisexual floral meristem.³³ A number of studies have shown that phytohormones play a pivotal role in the process of the selective arrest or abortion of pistils or stamens in female and male flowers, respectively.^14,15,26 The availability of GA plays an essential role in the expression of feminizing An1 (Anther earl) and D (Dwarf) genes in maize flowers.^33,34 In cucumber, ethylene is the key hormone involved in sex determination.^16,35 The expression of two genes, CS-ACS1 and CS-ACS2, encoding the ethylene biosynthetic enzymes (1-aminocyclopropane-1-carboxylic acid synthase), correlated with sexual phenotypes.³⁵ Recently, Martin & others³⁶ proposed an integrated model of sex determination in melon plants in which the andro-monoecious gene CmACS-7, encoding an ethylene biosynthesis enzyme,³⁷ and the gynoecious gene CmWIP1, encoding a zinc-finger transcription factor, interact to control the development of male, female, and hermaphrodite flowers. The expression of the pistil repressor CmWIP1 causes the arrest of carpel development and the repression of the expression of the stamen repressor CmACS-7, leading to the formation of male flowers. The inactivation of CmWIP1 by promoter hyper methylation, which also indirectly leads to the activation of CmACS-7, permits the development of female flowers. Hermaphrodite flowers resulted from CmWIP1 repression and the presence of a non functional CmACS-7 gene.^36,37 These results demonstrated that genes encoding metabolic enzymes for different phyto-hormones and the related transcription factors play important roles in the sex determination of various plant species. Thus by the application of plant growth regulators particularly, BA and GA₃ on the Jatropha curcas plantation, its production can be enhanced from 2 to 3.5 fold depending upon their concentration. 50mg of GA₃ and 100mg of BA were found to enhance the fruit and seed yield significantly and hence the availability of raw material for biodiesel production.^38–42

The possible reason of failure of Jatropha as a bio-fuel crop is due to mass plantation of low yielding germplasm which resulted in low yield per plant and per hectare. Therefore, application of PBRs at right time and in optimum quantity (50mg of GA₃ and 100mg of BA) can result in higher yield (more than one kg per plant & <1 ton per hectare) which will surely enhances the Jatropha production and may result in its consideration further as a viable source for Bio-diesel in future because this plant has potential due to low water requirement and its ability to grow in marginal lands and is also not consumed by stray and wild animals.

We thank the DRDO, Min. of Defence, Govt. of India for financial support and technical and scientific staff of DIBER is acknowledged for providing help during the preparation of this manuscript.

Author declares there is no conflict of interest towards the manuscript.

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