Variation among family of Pongamia pinnata (L.) Pierre for oil content and seedling growth

Pongamia pinnata (L.) Pierre is a multipurpose tree species that produces non-edible seeds. Pongamia oil has been recognized as a viable source of oil for the burgeoning biofuel industry. The economic feasibility of Pongamia depends on the oil content of the seed. Meanwhile, information on variation among families (parent tree) of seedlings grown is also necessary to increase plant productivity besides the oil content. This study aims to determine the variation among families of Pongamia oil content and seedling growth (generative and vegetative propagation). The seeds were analysed using 48 families for oil content analysis by solvent extraction, 50 families for seedling growth analysis (generative), and 19 families for vegetative. The result showed a significant variation in crude oil content among the families. Oil production varied from 26.61 to 44.68%. Variations among families were also found in seedling growth performance for both generative and vegetative propagation, with an average survival rate up to 75%. The information is essential for the tree improvement program to produce genetically improved seeds of Pongamia for biofuel and land restoration in Indonesia.


Introduction
Pongamia pinnata (Pongamia) is a fast-growing, medium-sized, evergreen/ briefly deciduous, and multipurpose tree species that produces non-edible seeds [1], [2].Traditionally, Pongamia has been employed in India and its neighboring countries as a source of traditional medicines, green manure, wood, animal feed, fuel, biopesticide, and fish poison [3].As a fuel, Pongamia is a potential resource for sustainable aviation fuel production due to the high oil content of its seeds [4].In recent years, Pongamia oil has been recognized as a viable source of oil for the burgeoning biofuel industry.
The economic feasibility of Pongamia depends on two factors.First, the oil content of the seed.Species with high oil content are desirable because they will give profit from biodiesel production.A study on Pongamia oil in Indonesia revealed that trees grown in Ujung Kulon National Park (TNUK), Banten, Province, produce bulk seed with a higher oil content (15.59%) than those in the province of East Java (14.49%) and West Java (13.13%) using a simple mechanical screw expeller press.However, the result was lower than in other countries using soxhlet extractor equipment (32-40% in India and 56% in the Philippines) [5]- [8].Second, information on seedlings' growth in the nursery to increase plant productivity.Pongamia can be cultivated by generative and vegetative with the percentage of rooted cuttings >90% [9], [10].The success of the development of Pongamia plantations is mainly 1315 (2024) 012066 IOP Publishing doi:10.1088/1755-1315/1315/1/012066 2 determined by the use of quality seeds (genetically, physically, and physiologically superior) that are available on time sufficiently, and adaptable to the environmental conditions in which they grow.Instead of generative propagation by seeds, cutting is an alternative vegetative propagation for the seeds of the species that are utilized as a source of biofuel materials [11].
The species selection and the source of the seeds within the species or source largely determine success in the establishment and productivity of forest plantation.Provenance is the original geographical area from which seeds or other propagules are obtained [12].Provenance from TNUK has a high oil content and 50 families have been collected as genetic material in the development of the Pongamia test [6].The families were prepared for generative and vegetative propagation materials in the nursery.Information on the growth performance of Pongamia seedlings from each family is the first step in preparing good and superior plant material for the development of the tested Pongamia.This study aims to determine variations among families of Pongamia for oil content and seedling growth (generative and vegetative propagation).The information on genetic variations is important for selection and tree improvement programs to produce genetically improved seeds of Pongamia for biofuel and land restoration in Indonesia [13]

Materials and methods
This study was carried out from 2019 to 2021.Seeds of Pongamia were collected from TNUK, Banten, Province, in 2019.The park is located on the western tip of Java and has a total area of 105.694, 46 ha (6°30'-6°52' LS, 102°02'-105°37') (Figure 1) [14].Oil content was extracted in the Bioenergy Laboratory, and plant propagation was conducted in the Center for Forest Biotechnology and Tree Improvement Research and Development Nursery, Sleman, Yogyakarta, from 2020 to 2021.Three replications of seed samples from 300-400 gr fruit pods were extracted manually for oil content analysis.Materials for crude oil analysis included n-hexane, water, paper filters, and seeds.Tools used to conduct the research included an analytical balance, dry seed blender, electric stove, distillation set, stone boiling, thermometer, glass funnel, and glass beaker [6].
Seed and stem cutting for propagation analysis were selected in similar sizes.For generative propagation, the seeds were extracted from the fruit pods, soaked in fungicide solution (Dithane M-45) for 1 hour, and planted in a polybag (15x10 cm) with a media mixture of soil and compost (3:1) at 1-2 cm in depth.The seedlings were watered daily in the morning and the plastic cover was opened gradually until the seedlings adapted well [15].For vegetative propagation, stem-cutting materials were taken from the 18-month seedlings.The seedlings were cut into 2-3 parts with an average length of 20 cm and 1-1.5 cm diameter.Stem cuttings were soaked in 50% rooting hormone solution (Root up) for 5 minutes.Stem cuttings are planted in polybags (8x13 cm) with sand media at 5 cm in depth.To maintain the humidity, stem cuttings were watered daily in the morning and covered with >80% sunblock plastic shade.

Oil Content
The oil from 48 families of Pongamia seeds was extracted with 2 methods: 1) mechanical pressing for bulk seed and 2) solvent extraction for bulk seed and individual seeds.The mechanical press method started with sun drying the seed samples for one to two days or in a cabinet dryer at 80 0 C to obtain dry seed with a moisture content of 8-12%.Before the screw press process, the ready samples were heated at 75-80 0 C for 5-10 minutes to 'loosen' the oil from the cake to ease the oil flow.The seeds are gradually inserted into the screw until raw oil and residue are released.This process can be repeated several times depending on the seed condition.Usually, raw oil can only be extracted after the resulting residue is completely dry.The oil content can then be quantified following a filtering process.
The process of the solvent method used 20-gram seeds grounded seeds.The fine seeds are wrapped using a paper filter and put into the Soxhlet apparatus assembled with a boiling flask containing 150 ml of n-hexane and boiling stones.Soxhlet and boiling flask are placed in a heater jacket containing ¾ water in a water bath, then heated on the stove.The temperature is kept stable in the range of 75-80 0 C. Oil extraction is carried out until all the oil is released from the seeds, marked with n-hexana dripping returning to its preprocess colour.The raw material was separated by a distillation process to obtain pure Pongamia oil.The Pongamia oil content was determined to examine the potential oil content variation.The formula for the oil content analysis followed the Indonesia National Standard on Calophyllum innophyllum as biodiesel raw material [16].Analysis of variance was applied to obtain the differences in oil content among families.

Generative propagation
Randomized Completely Block Design was conducted using 50 families, 10 seedlings in each family with 4 replicates.The total of seedlings used in this study was 2000.Seedling survival rate, growth performance (height, diameter, leave number), and sturdiness ratio were measured monthly up to 5 months after sowing.Analysis of variance was used to find out differences among families.Correlation among characters was analyzed by Pearson Correlation Analyses [ 1 ] .

Vegetative propagation
The study was arranged in a Randomized Block Design using 19 families, 5 individuals per family, and 4 replicates with a total sample of 380 stem cuttings.The seedling survival rate of stem cuttings, sprouting percentage, rooting percentage, and number of sprouts were measured monthly for up to 2 months.Analysis of variance was used to find out differences among families [2].

Oil content of Pongamia oil
Seeds from the same provenance with different oil-extracting methods produce different extracted oils.The solvent method extracts more oil than mechanical press, varying from 27.34% to 39.26% than mechanical press, ranging from 15.9% to 19.60% (Table 1).Thick and wet pressing seed residue waste containing oil was generated from the mechanical press method, and maximum oil yield was not obtained.Generally, conditions that affect the amount of oil yield are material, method, temperature, and pressure [17].In solvent method extraction processes, the oilseeds are washed with n-Hexane, its attributes such as simple recovery, non-polar nature, and high selectivity to the solvent [18].Moreover, the solvent method has advantages:1) maximum oil recovery, 2) lesser working cost, 3) production meets demand, 4) the extracted oil is low in sedimentation, and 5) solvent loss is low [19].The disadvantages of this method are that it needs longer and smaller production scale.To process oil content needed up to 4-6 hours, same with green betel leaf for hand sanitiser [20].However, the solvent method has been proven to produce high oil content and has developed in many countries, including Indonesia, which has many resources.The analysis of Pongamia oil from 48 families was significant (F= 4.00, P= <0.0001), indicating a high potential for increasing gains in the breeding program.Furthermore, the variation in seed of content from 48 families mean, and rank is presented in Table 2.The average oil content of the seed from these parents is 34.01%ranging from 26.61% to 44.68%.This result shows the potential of Pongamia oil content, which can reach 40% and was also higher than in other countries.Pongamia oil content varied from 26.65% to 33.12% in Thailand, from 33.31% to 39.01% in India, from 28.18% to 41.32% in the Philippines, and from 36.7% to 37.74% in Australia [8], [21]- [23].
There were 7 families from TNUK, Banten Province, performed best in oil content (16, 7, 44, 11, 10, 38, and 8), which reached >40% from 48 families observed.It becomes important information for the success of a tree improvement program on breeding strategy and the selection practices used to increase the expected genetic gain.Natural selection without human intervention will have less chance to increase genetic gain for randomly crossing without selecting the desired phenotypic characters.Tree breeding is conducted following the selection, and the offspring are used for a further selection of resources for the next generation's improvement [24].

Generative propagation
The average Pongamia seedlings survival rate is 84.60%.Of the 50 families observed, 48 have a survival rate of >60%, varying from 26.70% to 100%.The results showed that the average of seedling height was 47.10 ± 9.72 cm, the stem diameter was 5.49 ± 0.69 mm, the average number of leaves was 15.41 ± 2.29, and the seedling sturdiness was 8.56 ± 1.34.From these observations, variation in growth was found among seed sources (families).Seedling height and diameter are the most common characteristics to asses seedling growth performance and quality indicators [25].
Pearson Correlation Analysis showed a strong positive correlation (0.68) between height and stem diameter.Good seedlings perform optimum growth in both characters.Seedling height and stem diameter were the standard criteria for assessing forest plant seedling quality (SNI 01-5006. .Environmental factors that affect seedling's growth are medium type and fertility, fertilization, shading, growth regulators, pest/disease attack, and water availability in the nursery [26], [27]. The result on growth distribution from 50 families shows that 88% of seedlings had a mean height >40 cm.90% of families had a mean diameter >5 mm, and 86% of the families had a mean number of leaves > 14 strands per individual seedling.Seedling sturdiness shows that 95% of families have an average toughness score greater than 6.These results are similar to growth in Carita, Banten at 3 months after sowing, which has an average height of 38.72 cm, average stem diameter of 3.62 mm, and average seedling sturdiness of 10.78 [28].The results of this study indicate that the seedlings did not meet the standard quality for plantation stocks.Field plantation preferably uses seedlings that have an average height of >50 cm [29].At 5 months after sowing, only about 36% of the seedlings per family have average size of >50 cm.Analysis of variance on 5-month-old Pongamia seedlings showed significant differences between families (P<0.01) in survival rate, height of seedlings, stem diameter, and seedlings sturdiness.Meanwhile, number of leaves was not significantly different (p>0.05)(Table 3).Significant influence of genetic factors on variations in seedling growth in nurseries has been reported, as well as in Acacia nilotica [30], Tamarindus indica [31], Dyospiros celebica [32], Antocephalus cadamba [33], and Pongamia pinnata [28].The results aligned with the analysis correlation which showed a positive IOP Publishing doi:10.1088/1755-1315/1315/1/0120666 correlation between seedling growth characters observed.
A highly significant variation (p<0.01) was found between families on height.Family 2 and 3 had the highest character value, whereas family 24 and 25 had the lowest value.The average growth of seedling stem diameter ranged from 4.7-6.5 mm in diameter, best shown by families 12 and 26, while the smallest are families 36 and 49.The average seedling sturdiness ranges from 6.1-10.2, with the smallest value of toughness shown by families 22, 24, and 25; meanwhile, the largest are families 3, 44, 45, and 47.The number of leaves ranges from 10.9-18.8strands, with most leaves found in families 3-26, while the lowest number was in family 49.

Vegetative propagation
Two-months-old stem cuttings showed an average survival rate of 75.84% from 19 families with a 50-95% distribution.The average percentage of sprouting cuttings was 75.26%, with a distribution ranging from 40-95%.Meanwhile, the rate of rooting cuttings ranged from 45-95%, and the number of shoots that grew ranged from 3-8 shoots.The results of these studies indicate that at the age of 2 months, stem cuttings have developed well in both the growth of shoots and roots.Of the 19 families, 14 families showed shoot and root growth rates of more than 60%.Another study with the same observation time reported a rooting percentage of 66.67% by the application of IBA 4.92 Mm [34].In general, the growth of stem cuttings in nurseries is strongly influenced by many factors, such as the parent tree, the degree of ageing of plant tissue, the planting medium, the humidity of the beds, and others [35]  Analysis of variance showed that at the age of 2 months old, there were significant variations in the growth of Pongamia stem cuttings between families in survival rate and percentage of shoots (Table 4).Meanwhile, the percentage of rootings and the number of nodes showed variations between families but no significant variations.The results of previous studies using juvenile plant tissue of shoots (shoot cuttings) obtained an average percentage of rootings of 91.11% [9].From the results of this study, it was found that for vegetative mass production of Pongamia seedlings, there was more potential to use juvenile plant tissue than mature cuttings of stems or branches.Pongamia propagation studies using stem cuttings have been widely reported.The best method for propagation through cuttings from mature trees, which resulted in 100% rooting, is as described: 1) cutting size: length -25 cm; circumference -1-1.5 cm; 2) treatment: 15 h of dipping of cut end in 800 ppm IBA; 3) conditions: 30 ± 2 •C; 70-80% relative humidity; 4) misting season: spring; based on the conclusion that the best season for root induction in the presence of exogenous auxin is the period when the cambium is already in active phase [36], [37].With that method, it is possible to commercially propagate Pongamia by stem cuttings with success ranging between 60%-80% if these recommendations are followed and thus help establish clonal plantations [38].Vegetative propagation is indispensable in tree repair programs, such as those required to establish clone seed gardens.Also, mass vegetative propagation of selected genotypes offers fast promise productivity gains.

Conclusion
Pongamia pinnata is a potential bioenergy species with a possible oil content of up to 44.68% using a solvent method.Influences on the production of Pongamia oil are genetic factors, extraction machines, and the extraction method.Based on the variation in oil content potential among trees, selection can increase the Pongamia oil productivity to produce genetically superior seeds to support tree improvement of Pongamia for biofuel.Increasing crop productivity can be achieved by generative and vegetative propagation.Variations among families were also found in seedling growth performance for both generative and vegetative propagation, with an average survival rate of up to 75%.The variations among families in Pongamia oil content and propagation will be used to establish progeny tests in several sites for biofuel and land restoration in Indonesia.

Table 1 .
Pongamia oil content from TNUK by two methods.

Table 2 .
Mean of oil content among 48 families.

Table 3 .
Analysis of variance of 5 months of Pongamia seedlings in the nursery.

Table 4 .
Analysis of variance of 2-month-old Pongamia stem cuttings in nursery