Intergeneric Hybrid from Jatropha curcas L. and Ricinus communis L.: Characterization and Polyploid Induction

Jatropha curcas L. (2n = 2× = 22) is increasingly attracting attention in the biodiesel industry for its oil. However, the cultivation of J. curcas L. is faced with numerous challenges unlike the cultivation of Ricinus communis L. (2n = 2× = 20), a closely related species. The generation of an intergeneric hybrid between J. curcas L. and R. communis L. was investigated. Intergeneric hybrids were produced by hand crossing. Immature embryos were rescued, in vitro, from the hybrid seeds and cultured on an enriched Murashige and Skoog (MS) medium for a month. The plantlets produced were grown in sterile peat moss in plastic pots and covered with polyethylene for 30 days, after which they were transferred into cement potted soil. The hybridity and the genuineness of the hybrids were successfully confirmed using randomly amplified polymorphic DNA (RAPD) markers. The number of branches, stem diameter, and leaf size of the F1 hybrids were similar to those of J. curcas L. while the plant height was similar to that of R. communis L. Young hybrids were treated with various concentrations (0%, 0.3%, 0.4%, and 0.5%) of colchicine to induce polyploids. The calli (JR6) treated with 0.3% colchicine recorded the highest tetraploid cell percentage (38.89%). A high tetraploid cell percentage (>50%) is significant in overcoming the problem of sterility after hybridization.


Introduction
Jatropha curcas L. (2n = 2× = 22), a perennial shrub, and Ricinus communis L. (2n = 2× = 20), a tropical and sub-tropical perennial shrub or temperate annual, are two very promising non-edible oilseed plants in the family Euphorbiaceae [1,2]. J. curcas L. has generated a lot of interest as a sustainable alternative source of seed oil for the production of biodiesel. On the other hand, R. communis L. is a source of medicinal and vegetable oil with numerous industrial and medical uses worldwide [3].
In spite of its potential in the biodiesel industry, commercial production of J. curcas L. is faced with numerous challenges including poor seed yield and non-synchronous flowering. Seed cultivation of J. curcas L. is often associated with heterozygosity, which affects the quality and quantity of the oil content. Propagation by cuttings also yields plants that are susceptible to drought and diseases [4]. Attempts to improve J. curcas L. through conventional breeding, radiation mutation, protoplast

Embryo Rescue of the Intergeneric F 1 Hybrids
Intergeneric F 1 hybrid fruits were harvested 40 days after pollination. The seeds were then excised and the surface immersed in ethanol (70%) for 30 s, followed by sterilization in 10% Clorox for 10 min and finally rinsed in sterile distilled water three times. Young embryos were removed from the seeds and cultured on a regeneration medium (RM). The composition of the RM (pH 5.7) included Murashige and Skoog (MS) medium [17] enriched with citric acid (30 mg/L), 6-benzylaminopurine (BA, 1 mg/L), indole-3-butyric acid (IBA, 0.25 mg/L), polyvinylpyrrolidone (PVP, 500 mg/L), sucrose (3% w/v), kinetin (Kn, 0.5 mg/L), and agar (0.7% w/v) [18]. The cultured embryos were incubated for four weeks at 25 • C under a 16 h light and 8 h dark photoperiod. After four weeks, the plantlets were grown in sterile peat moss in small plastic pots and covered with polyethylene bags for 30 days. The young seedlings that developed after 30 days were transferred into cement potted soil in the field.

Morphological Characterization
The morphological characteristics of both the parental and intergeneric F 1 hybrid plants were evaluated according to [19,20]. The evaluated characteristics were stem diameter (mm), plant height (mm), total number of branches per plant, number of female and male flowers, seed size (length and width, mm), and leaf size (length and width, mm).

DNA Isolation
Genomic DNA was extracted from fresh young leaves and purified using Omega Plant DNA Kit (D3485-02, Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's protocol. The extracted DNA was quantified using the UV spectrophotometer (Analytik Jena Specord 40, Analytik Jena AG, Jena, Germany) at the absorbance ratio of 260/280 nm. The DNA concentration was adjusted to 100 ng/mL.

Randomly Amplified Polymorphic DNA (RAPD) Marker Analysis
A total of 46 random primers (Operon Technology Inc., Alameda, CA, USA) were screened against the template DNA. A reaction volume of 25 µL containing template DNA (100 ng), 1× PCR buffer (with 15 mM Mg 2+ ), 2 mM MgCl 2 , 0.2 mM dNTP mix (Fermentus Pvt. Ltd., Bangalore, India), single primer (0.8 µM), and Taq DNA polymerase (0.5 U) was used. For the initial denaturation, the thermocycler (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA, USA) was set at 94 • C for 3 min, followed by 45 cycles consisting of 1 min denaturing at 94 • C, annealing for 1 min at 37 • C, 2 min of extension at 72 • C and finally, 7 min of extension at 72 • C. The amplification products (10 µL) were electrophoresed through 1.5% (w/v) agarose gel (Research Organics Inc., Cleveland, OH, USA) in 1× Tris-acetate-EDTA (TAE) at 100 V for 30 min. This was followed by staining with 0.5 µg/mL ethidium bromide. The resulting fragments were observed and photographed under a UV transilluminator using a gel documentation system. The banding patterns of the intergeneric F 1 hybrid and their parents for a specific primer were observed and compared. The reproducibility of the RAPD patterns was confirmed by repeating the PCR analysis [21][22][23]. RAPD markers have been extensively studied and successfully used for the identification and confirmation of F 1 hybrids in plant breeding [14]. The RAPD analysis is technically simple, cheap, requires little amount of DNA, produces large number of polymorphic markers, and does not require sequence information [15].

Statistical Analysis
The data collected on growth characteristics were subjected to one-way analysis of variance (ANOVA) using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Treatment means were compared by Tukey's test at a 5% significance level. The average values and their corresponding standard deviations were presented.

Sexual Intergeneric Hybridization and Embryo Rescue
The effect of GA 3 , NAA, and calcium-boron on fruit set, seed set, and embryo development of the cross and reciprocal cross between the female parent, J. curcas L., and the male parent, R. communis L. (Figure 1), were observed and are shown in Table 1. Among the treatments, the intergeneric F 1 hybrid treated with 0.1% (v/v) calcium-boron had a high percentage of fruit set (40%) and 100% seedling survival. However, the percentage of seeds set as a result of the application of 25 mg/L GA 3 and 0.1% (v/v) calcium-boron on the stigma was the same (83%). The immature embryos rescued by in vitro culture developed into whole plants ( Figure 2). Of the 100 intergeneric F 1 hybrid embryos rescued, 17 regenerated into plantlets, from which eight developed into plants in the field. In contrast, the reciprocal crossing between J. curcas L. and R. communis L. was not successful. Embryo abortion was observed 20 days after pollination ( Figure 3). The seed capsule was found to be empty with no kernel ( Figure 3B).

Identification of Intergeneric F 1 Hybrid Using Morphological Characteristics
Morphologically, the F 1 hybrid plants were compared to the parents ( Table 2). The results showed that the fruit, seed, and stem of the F 1 hybrids were similar to those of J. curcas L. (Figures 1-3). The leaf size of the F 1 hybrid plants was significantly (p < 0.05) lower when compared to that of R. communis L. but similar (p > 0.05) to that of J. curcas L. However, the number of branches per plant and stem diameter of the F 1 hybrid plants were significantly (p < 0.05) higher than those of R. communis L. but similar (p > 0.05) to those of J. curcas L. The number of female and male flowers of the F 1 hybrid plants was significantly lower (p < 0.05) when compared to that of the parent plants. In addition, the plant height of the F 1 hybrid plant was significantly (p < 0.05) lower when compared to that of J. curcas L. but similar (p > 0.05) to that of R. communis L. (Table 2). Finally, the seed size of the F 1 hybrid plants was intermediate between the parent plants ( Figure 4). Generally, the F 1 hybrids exhibited morphological characteristics of both parents.

Identification of Intergeneric F1 Hybrid Using Morphological Characteristics
Morphologically, the F1 hybrid plants were compared to the parents ( Table 2). The results showed that the fruit, seed, and stem of the F1 hybrids were similar to those of J. curcas L. ( Figure 1 Figure 2 Figure 3). The leaf size of the F1 hybrid plants was significantly (p < 0.05) lower when compared to that of R. communis L. but similar (p > 0.05) to that of J. curcas L. However, the number of branches per plant and stem diameter of the F1 hybrid plants were significantly (p < 0.05) higher than those of R. communis L. but similar (p > 0.05) to those of J. curcas L. The number of female and male flowers of the F1 hybrid plants was significantly lower (p < 0.05) when compared to that of the parent plants. In addition, the plant height of the F1 hybrid plant was significantly (p < 0.05) lower when compared to that of J. curcas L. but similar (p > 0.05) to that of R. communis L. (Table 2). Finally, the seed size of the F1 hybrid plants was intermediate between the parent plants ( Figure 4). Generally, the F1 hybrids exhibited morphological characteristics of both parents.

Identification of Intergeneric F1 Hybrid Using Morphological Characteristics
Morphologically, the F1 hybrid plants were compared to the parents ( Table 2). The results showed that the fruit, seed, and stem of the F1 hybrids were similar to those of J. curcas L. ( Figure 1 Figure 2 Figure 3). The leaf size of the F1 hybrid plants was significantly (p < 0.05) lower when compared to that of R. communis L. but similar (p > 0.05) to that of J. curcas L. However, the number of branches per plant and stem diameter of the F1 hybrid plants were significantly (p < 0.05) higher than those of R. communis L. but similar (p > 0.05) to those of J. curcas L. The number of female and male flowers of the F1 hybrid plants was significantly lower (p < 0.05) when compared to that of the parent plants. In addition, the plant height of the F1 hybrid plant was significantly (p < 0.05) lower when compared to that of J. curcas L. but similar (p > 0.05) to that of R. communis L. (Table 2). Finally, the seed size of the F1 hybrid plants was intermediate between the parent plants ( Figure 4). Generally, the F1 hybrids exhibited morphological characteristics of both parents.

Identification of Intergeneric F 1 Hybrid Using RAPD Analysis
RAPD analysis of genomic DNA was performed to confirm the hybridity of the intergeneric F 1 hybrids. Initially, 45 primers were screened for polymorphism in the parents. From these 45, only 10 distinguished the parents. The 10 primers (OPA-01-OPA-10) were used to amplify DNA from the F 1 hybrids along with the parents. Out of the 10 primers, only one (OPA-07) generated polymorphic PCR bands of both parents in the F 1 hybrids of J. curcas L. and R. communis L. (Figure 5). The clear and specific amplified DNA fragments of J. curcas L. were 400 and 600 bp, whereas those of R. communis L. were 300, 400, 800, 900, and 1200 bp. Similarly, the hybrids showed specific bands of J. curcas L. and R. communis L. The F 1 hybrid JR6 showed the clearest specific bands of both J. curcas L. (400 and 600 bp) and R. communis L. (300, 400, 800, 900, and 1200 bp) ( Figure 5). RAPD analysis of genomic DNA was performed to confirm the hybridity of the intergeneric F1 hybrids. Initially, 45 primers were screened for polymorphism in the parents. From these 45, only 10 distinguished the parents. The 10 primers (OPA-01-OPA-10) were used to amplify DNA from the F1 hybrids along with the parents. Out of the 10 primers, only one (OPA-07) generated polymorphic PCR bands of both parents in the F1 hybrids of J. curcas L. and R. communis L. (Figure 5). The clear and specific amplified DNA fragments of J. curcas L. were 400 and 600 bp, whereas those of R. communis L. were 300, 400, 800, 900, and 1200 bp. Similarly, the hybrids showed specific bands of J. curcas L. and R. communis L. The F1 hybrid JR6 showed the clearest specific bands of both J. curcas L. (400 and 600 bp) and R. communis L. (300, 400, 800, 900, and 1200 bp) ( Figure 5).

Induction of Polyploids in the Intergeneric Hybrids
The calli JR1 and JR6 successfully responded to the colchicine application in vitro ( Table 3). The ploidy percentage was determined from the peak area of the histogram of the relative fluorescence intensity of nuclei of each plant using flow cytometry. The diploid J. curcas L. showed one clear peak at channel 200 ( Figure 6A) while R. communis L. presented peaks at channel 100 and 200 ( Figure 6B). The highest tetraploid percentage (38.89%) was observed in the F1 hybrid JR6 after treatment with 0.3% colchicine for three days ( Figure 6C).

Induction of Polyploids in the Intergeneric Hybrids
The calli JR1 and JR6 successfully responded to the colchicine application in vitro ( Table 3). The ploidy percentage was determined from the peak area of the histogram of the relative fluorescence intensity of nuclei of each plant using flow cytometry. The diploid J. curcas L. showed one clear peak at channel 200 ( Figure 6A) while R. communis L. presented peaks at channel 100 and 200 ( Figure 6B). The highest tetraploid percentage (38.89%) was observed in the F 1 hybrid JR6 after treatment with 0.3% colchicine for three days ( Figure 6C).

Discussion
Intergeneric hybridization is a very useful technique for improving crop species worldwide [16]. It has been used to develop several F1 hybrid plants with genetic variability from the parent plants [16,24,25]. However, one major barrier to intergeneric hybridization is the problem of very low or no seed set [26]. Tang [25], for example, reported the absence of seeds in F1 hybrids after an intergeneric hybridization between Dendranthema nankingense and Tanacetum vulgare. Zhao [24] also reported only 5% seed set after an intergeneric cross between Dendranthema × grandiflorum "Aoyunhuoju" and Ajania pacifica. To overcome this barrier, several chemical formulations have been used by different researches to enhance seed set. Dinesh [27] reported 13.1% seed set per fruit after application of 5% sucrose to the stigma of Vasconcellea cauliflora during the intergeneric crossing between Carica papaya var. Surya and V. cauliflora. Jayavalli [28] also reported an enhancement in both fruit and seed set after application of 5% sucrose, 0.5% boron + 5% sucrose, and 0.5% CaCl2 + 5% sucrose during the intergeneric hybridization of C. papaya and V. cauliflora. Application of 0.1% calcium-boron to the stigma of the female parent plant (J. curcas L.) in the current study resulted in 100% intergeneric F1 hybrid seedling survival with high fruit set and seed set (Table 1). Treating the stigma of the female parent plant with GA3, NAA, and calcium-boron may promote early pollen germination, increase the period of pollination, and improve the growth of the pollen tube because these compounds influence fertilization and increase cell division during fertilization [29].
The F1 hybrid seeds in this study however were shrunken with slimmed embryos (Figure 1), an

Discussion
Intergeneric hybridization is a very useful technique for improving crop species worldwide [16]. It has been used to develop several F 1 hybrid plants with genetic variability from the parent plants [16,24,25]. However, one major barrier to intergeneric hybridization is the problem of very low or no seed set [26]. Tang [25], for example, reported the absence of seeds in F 1 hybrids after an intergeneric hybridization between Dendranthema nankingense and Tanacetum vulgare. Zhao [24] also reported only 5% seed set after an intergeneric cross between Dendranthema × grandiflorum "Aoyunhuoju" and Ajania pacifica. To overcome this barrier, several chemical formulations have been used by different researches to enhance seed set. Dinesh [27] reported 13.1% seed set per fruit after application of 5% sucrose to the stigma of Vasconcellea cauliflora during the intergeneric crossing between Carica papaya var. Surya and V. cauliflora. Jayavalli [28] also reported an enhancement in both fruit and seed set after application of 5% sucrose, 0.5% boron + 5% sucrose, and 0.5% CaCl 2 + 5% sucrose during the intergeneric hybridization of C. papaya and V. cauliflora. Application of 0.1% calcium-boron to the stigma of the female parent plant (J. curcas L.) in the current study resulted in 100% intergeneric F 1 hybrid seedling survival with high fruit set and seed set (Table 1). Treating the stigma of the female parent plant with GA 3 , NAA, and calcium-boron may promote early pollen germination, increase the period of pollination, and improve the growth of the pollen tube because these compounds influence fertilization and increase cell division during fertilization [29].
The F 1 hybrid seeds in this study however were shrunken with slimmed embryos (Figure 1), an observation that has been reported in other research [14,30]. Interspecific or intergeneric hybridization is limited by postzygotic incompatibilities including embryo abortion and degeneration, resulting in a decrease in fertility. However, postzygotic incompatibilities can be overcome by the use of embryo rescue techniques [31]. Embryo rescue has been successfully used to overcome postzygotic incompatibilities following interspecific or intergeneric hybridization of a wide range of plant species, subsequently leading to the development F 1 hybrids [14,30,32]. An average regeneration efficiency of only 17% was recorded after the embryo rescue of F 1 hybrid embryos in the current study, which may be due to several factors including the media composition and the developmental stage of the embryo [31]. Rodrangboon [14] reported an average in vitro embryo regeneration efficiency of 25.6% for F 1 hybrid (Oryza sativa × Oryza officinalis) embryos obtained 7-10 days after pollination. The efficiency however increased to 55.6% for embryos obtained 11-14 days after pollination. In vitro culturing of F 1 hybrid (Brachiaria ruziziensis × Brachiaria decumbens or Brachiaria brizantha) of 9-12 days old embryos yielded over 80% regeneration efficiency unlike approximately 35% for 7-8 days old embryos [30]. Excising the F 1 hybrid embryos at the optimal developmental stage and adjusting the composition of the RM as well as the culture conditions in the current study may enhance the efficiency of regeneration [31].
The F 1 hybrid plants showed morphological resemblance to both parents, a clear indication that they are from the intergeneric crossing, however, hybridity was confirmed using a RAPD marker. RAPD analysis is one of the most commonly used molecular techniques for the study of genetic diversity [21]. It has been successfully used to confirm hybridity in F 1 hybrids including Passiflora [11], Solanum [33], Capsicum [34], and Centaurium [13]. The clear expression of the specific bands of both parents in the F 1 hybrid JR6 ( Figure 5) is a confirmation of hybridity and the authenticity of this particular hybrid plant in the current study. This result confirms the successful production of an intergeneric F 1 hybrid from the female parent, J. curcas L., and the male parent, R. communis L.
Sterility of F 1 hybrids has often been observed in intergeneric hybridization [14]. Polyploidization in F 1 hybrids is a process that can help to overcome sterility. Polyploids can be induced in the F 1 hybrids through the application of various antimitotic agents, including colchicine, trifluralin, oryzalin, and amiprophos-methyl [6]. However, polyploidy induction has mainly been accomplished by the use of colchicine. Induction of polyploids through the application of colchicine has been reported by several researchers [35][36][37]. In the current study, attempts were made to double the chromosome set of the F 1 hybrid through the application of colchicine. However, the tetraploidy percentage was low. The concentration and duration of colchicine application, among other factors, plays a key role in the success of polyploidy induction [38]. Polyploidy has been successfully induced in Lychnis senno with the application of colchicine concentrations as low as 0.00001% [35] and in Chaenomeles japonica with a very high concentration of 1.5% [39]. Several studies have shown that the application of high colchicine concentrations for lengthy periods is effective in the successful induction of polyploids [36,40,41]. However, very high colchicine concentrations for a longer time have been reported by several researches to be very harmful to the explants [35,38,39]. Expanding the range of colchicine concentrations and varying its application period, with a balance between survival and successful polyploidy induction in mind, may help to improve the polyploidization process in the current study. Castro [37] recommended the selection of seedlings at the ideal stage of development for the polyploidization process after reporting the efficient induction of polyploidy in younger seedlings when compared to older ones. Unfortunately, this factor has not been well exploited by most researchers. The calli age and stage of development may also be exploited to enhance the polyploidization process in the current study.

Conclusions
An intergeneric F 1 hybrid from the female parent, J. curcas L., and the male parent, R. communis L., was produced. The hybridity and authenticity of the F 1 hybrid was successfully confirmed. However, a low tetraploidy percentage was recorded. Expanding the range of colchicine concentrations and varying its application period as well as exploiting the calli age and stage of development may help to enhance the polyploidization process. The results in the current study form a strong basis for further research to enhance intergeneric hybrid production from J. curcas L. and R. communis L.