Towards the fourth decade of IOPRI’s oil palm clones: Upcoming new variety

Experience of IOPRI on oil palm tissue culture would be four decades in 2024. The first decade was theperiod of oil palm clone development and field testing. The second decade was a setback for oil palm research because of a high percentage of oil palm clone abnormalities in the field. Moving on from these experiences, tissueculture systems such as: including improving the protocol, controlling the duration of callus induction, delimitating the number of subcultures, and building of oil palm clones database, were managed to be improved. In this way, alow abnormality level is under control and each clone is traceable. It is also noted that the oil palm clone provides20-30% higher production than that derived from DxP crossing seed due to the more uniform growth of generatedplants. Besides the protocol, ortet selection is also key to improvement. Ortets are elite plants from selected progeny testing or re-clone of the progeny testing. Molecular analysis is applied as a control of genetic variation possibility, which may occur in a particular phase due to specific stimuli. However, until the present time, a clonevariety has not been released yet. Therefore, in the fourth decade, mass production is expected and oil palm clonevarieties will officially be released and commercialized


Introduction
In the 1980s, the area of Indonesian oil palm plantations was approximately 200,000 hectares.Over the next decade, in the 1990s, the area of oil palm plantations expanded rapidly and reached around 1.7 million hectares.By the 2000s, the total area of Indonesian oil palm plantations had further increased to approximately 4.15 million hectares.This significant growth in oil palm plantations reflects the expansion of the industry and its importance in Indonesia's agricultural sector.The significant increase in the area continued to occur until it reached 7.82 million ha in 2010 and currently has reached 14.62 million ha [1,2].There has been a rise of 12.92 million ha in the last 40 yearsor an increase of 88.37%.With this area, until 2021, Indonesia had produced high Crude Palm Oil (CPO) production, reaching 45.1 million tons.Therefore, the potential for developing oil palm is verypromising for palm oil business players.

The first decade of the 1990s: Establishment and development of oil palm clones
The establishment of laboratory facilities by IOPRI in 1984 and the subsequent research on oilpalm tissue culture in 1985 demonstrate the institute's commitment to advancing the field.By adopting technology from CIRAD-CP (Center for International Cooperation in Agricultural Research for Development -Oil Palm Research Unit), IOPRI was able to leverage existing expertise and knowledge to kickstart its research efforts [9].The studies conducted by IOPRI on oil palmtissue culture paved the way for the development of the institute's oil palm clones.These clones were first planted in Jambi Bah Plantation, PTP Nusantara III in 1987, followed by Cot Girek Plantation, PTPNusantara I in 1988, and Seberang Oil Palm Plantation, PTP Nusantara II in 1989.Other tests in this eraincluded the Tinjowan plantation, PTP Nusantara IV and Sei Pagar and Sei Berlian Estate, PTP Nusantara V [10,11].Furthermore, clone planting was carried out in several areas including Aceh, Sumatra, Riau, Jambi, West Java, and Kalimantan.The results of the initial IOPRI clonetesting showed consistent results with good clone productivity which was indicated by a high percentageratio of DxP clones to oil palm seeds ranging from 120-130% [12].
The results of observations of other laboratory clones from several institutions also showed similar things.After 15 years, the IDEFOR-DPO laboratory produced performance data on tissue culture results.The experimental results conducted in Ivory Coast in 1997, as reported by [13], demonstrate the potential benefits of using oil palm clones for increased production.In the experiment,16 clones were compared to the control, which consisted of L2T x D10D sprout crosses.The results showed that these clones significantly increased production ranging from 10% to 54%.This indicates that the use of clones can lead to improved productivity compared to standard DxP materials.Furthermore, the study also evaluated the performance of 15 clones produced by Socfindo, IOPRI and ORSTOM.These clones exhibited superior yields compared to the control, with increases ranging from8% to 44%.The findings reported by [4] indicate that the testing of 14 clones resulted in significantly higher production yields compared to the standard DxP (Deli Dura x Pisifera) materials in oil palm cultivation.According to their study, the average clone production yield was 35%higher than that of the standard DxP.The clones exhibited an average yield of 8.2 tons per hectare (t/ha),while the standard DxP had an average yield of 6.2 t/ha.
Problems in the form of somaclonal variation or clonal abnormalities were found in several testsin the early decades.Symptoms of abnormality are usually seen from the generative phase, like in flowers and fruit.This is very detrimental because it can reduce plant productivity.At the end of the first decade, more and more tests showed somaclonal variation as seen from the abnormal floral of the clones.This did not only occur in clone testing in Indonesia but also in several laboratories conductingtests in various countries.Due to these abnormality constraints, the commercialization of oil palm clonesin the early decades experienced some obstacles.

Second decade, the 2000s: Setback of oil palm research due to somaclonal variation
In the second decade, around the 2000s, there was a setback in oil palm clones due to abnormalities in clone field testing.In this year's range, the percentage of abnormalities increased quitehigh (Table 3).This problem arose in line with the continuous-ongoing research and clonal multiplication of oil palm in various laboratories in Indonesia.Clonal abnormalities caused a decrease in plant productivity by up to more than 50% of its production potential at high clone severity levels.The greater the clone abnormality, the lower the productivity of the clone in the field.For instance, testdata from various clone test farms showed that plantations with an abnormality percentage of 0% produced the highest clone productivity.Meanwhile, plantations with various and high percentages of abnormalities showed very low clone productivity (Figure 6a).Floral abnormalities in oil palm clones, specifically the occurrence of mantled flowering, have been attributed to epigenetic factors.Epigenetics refer to gene expression or cellular phenotype changes that do not involve alterations in the underlyingDNA sequence.These changes can be influenced by various factors, such as environmental conditions or developmental processes [7,[14][15][16][17][18].Many studies on clonal floral abnormalities have been carried out including by [7] and their results showed differences in DNA methylation found innormal and abnormal embryogenic calluses.The differences observed in the location and number of restriction cycles between normal and abnormal fruit indicate potential variations in the DNA sequencesor epigenetic modifications associated with these different phenotypes.
Observation of genotypic abnormalities is carried out through molecular pathways because only this technology is capable of investigating differences in normal and abnormal clone DNA.Several studies have been conducted to look for DNA markers that cause abnormalities in oil palm clones.[7] conducted a study on screening 27 sets of cDNA in oil palm using the RFLP (Restriction Fragment Length Polymorphisms) method to determine the presence of DNA methylation using nodular compact and fastgrowing calli as samples.Moreover, the enzyme isometrics are MspI and HpaII.The results showed that there were only two probes, namely CPHO62 and CPHO63 which resulted in genotypic differences in DNA methylation between normal and abnormal embryonic calluses.
According to [19], in plants that produce male and female mantle flowers, theplants do not produce fruit due to an abortion called the sterile mantle.These plants will never recover to typical floral plants.
The sterile mantle is usually classified as a heavy mantle because it does not yield.According to the study by [20], the light mantle will experience a decrease in the percentage of abnormalities faster than the heavy mantle.Light mantle on the early flowering (plant twoyears old) experienced a decrease in abnormality by almost 80% at five years of age and more than 95%at nine years.However, for the heavy mantle, from the early flowering at the age of 8 years, the abnormality only decreased by 50% (Figure 1).This shows that light mantle still gives quite good results,considering that the productivity of the clones is 20-30% higher than that of oil palm from seedlings.
Recent research regarding abnormalities in oil palm clones was published by [18], showing that the 3rd transposon in the KARMA section is closely related to the abnormal phenotypic in oil palm.
Research by [21] indicated that DNA methylation in the class Bgene (EgDEF1) activated the occurrence of TE which is located in the 5th intron of the upstream promoter.Research on mantle fruit that is currently associated with the MADS-Box homeotic gene is related to the formation of flowering organs, especially those related to carpel formation where hereditary genes are inherited, namely the expression of the EgDEF and EgGLO genes [22].

Third decade, the 2010s: Improvement, innovation and implementation of a new oil palm tissue culture system
In the third decade, in the 2010s, with various experiences from tissue culture research and clonefield testing, IOPRI conducted research and innovation from various aspects in an effort to reduce the level of abnormalities in the field.Some of the efforts made included:

Identifying processes in the Laboratory that can cause clone abnormalities
The abnormality of the clones produced was influenced by the time the culture was in the laboratory, either during the induction process or the number of sub-cultures performed.The research conducted by [24] and published by IOPRI focused on the effect of sub-culturing in the embryonic phase on the formation of mantle fruit bunches in oil palm clones.Theresults of the study indicated that the number of sub-cultures during the embryonic phase impacted the occurrence of mantle fruit bunches.By manipulating the number of sub-cultures, the researchers could observe a correlation between the subculturing process and the manifestation of the mantle phenotype.The number of subcultures carried out was linear with thepercentage of clone abnormalities found.The lowest percentage of clonal abnormality was only 1.28%, and if subculture was carried out 1-5 times, the percentage increased successively, namely 2.95%, 3.74%, and 3.83% in sub-culture 6-10 times, 11-15 times, and 16-20 times.Meanwhile, in more than 20 sub-cultures, the abnormality increased rapidly to 18.15% (Table 1).The publication stated that clone abnormalities were classified as low if the percentage was equal to or less than 5%.
Another study from IOPRI was regarding the limitation of the length of time for callus formation which affected the percentage of abnormalities in the field.The result showed that the higher the percentage of clonal abnormalities observed if the callus formation time is longer.Thepercentage of abnormalities was below 2% with a duration of callus formation of less than 200 days (Table 2).This is a reasonably long study because the data analysed resulted from research and innovation by IOPRI with a new database-based culture system and culture restrictions that started in 2009.In addition, IOPRI has also identified various types of callus produced in the laboratory which may affect abnormalities in the field.[26] classified various types of induced oil palm callus at IOPRI based on the shape and texture of the callus.Based on the shape, the calli are divided into filiform, granular, and pseudo-root calli.Meanwhile, based on the texture, calli are divided into an aggregate, aqueous, and friable callus.From various publications, it is stated that the classification of callus is into 2 groups, namely fast-growing andnodular compact calli.Hundred per cent of heavy mantled will be produced by fast-growing calli [27][28][29].
Another identification is in the vegetative stage of plantlets to seedlings, which causes vegetative abnormalities in the clones.[30] grouped abnormal clones vegetatively into plantlet, pre-nursery, and main nursery phases.In the plantlet phase, plantlet abnormalities were rosette, dry tips, erect, pseudo plantlets, less than four leaves, white tips, dwarf, curved, terminal inflorescence, and yellowish.During the pre-nursery and main nursery phases, abnormal seedlings were in the form of curved, rosette, dry tips, erect, dwarf seedlings, and pseudo plantlets.This abnormality will also have an impact on the performance of the clonesin the field if the selection is not carried out at each stage before planting in the field.One of theabnormal results of clones that were not selected during planting was erect midrib found in severalIOPRI clone experimental gardens in North Sumatra and South Sumatra.

Traceable database management
Since 2009, IOPRI has built a database system to support tissue culture systems in laboratories with better records and traceability.The database was created by integrating systems andprocesses in each phase of culture, starting from the collection of ortets to the production of plantlets.This system has undergone two-time improvement since it was implemented.All IOPRIclones that have gone through a traceable database process are known as FTC clones or fully traceable clones [31,32].The database program consists of master data, ortet identification, entire laboratory processes, and a summary of all data.The clonesproduced were tested in the field with the first planting in 2013 and continued with the next test in 2015 [33].

Various research and innovation.
Research to improve the culture method of each phase that has been conducted by IOPRI includesthe callus phase with callus type classification to determine callus quality [26], and cell suspension to increase the multiplication of young oil palm embryos quickly [34].Research on the status of phytohormones in the source of oil palm ortets aimed to find out how much endogenous hormones are contained in ortets so thatit can be used as a basis for determining how much exogenous hormones or growth regulators (ZPT) need to be added in callus formation [35].It is expected this study is carried out regarding the length of time of culture, age, and leaf number at the ortet source on callus formation [36].Knowing the effect of time on callus formation can determinethe optimal time for callus formation.In addition, the research continued to field testing to determine the effect of callus formation time on clone abnormalities in the field [25].Vegetative grouping of normal and abnormal clones was carried out in the plantlet, pre-nursery and main nursery phases [30], and field testing was carried out to determine the performance of clones in the field from an early age with routine monitoring and evaluation [8].In addition, research for efficiency in clone production in the laboratory was also carried out, including agar substitution [37], optimizing the number of plantletsand adding NAA in plantlet growth [38], and research related to lightintensity and filters on development of oil palm culture [39].Other research for plantlet distribution was by optimization of acclimatization research [40], sending of plantlets [41] and clone seeds [42], use of natural light during the hardening period [43] immersion of plantlets in water as pre-aclimatization of plantlets [44], optimization of sterilization protocols to reduce culture contamination [45], and standardization of seedling quality [46].

Improvement by DNA analysis
DNA analysis is used as a guarantee for the identity of clones grown according to their ortet source and to see the uniformity and gene stability of the clones produced.The DNA fingerprinting process typically involves extracting DNA from the leaf samples, amplifying specific regions of the DNA using techniques such as Simple Sequence Repeat Anchored Polymerase Chain Reaction (SSR-PCR), and separating the resulting DNA fragments using gel electrophoresis or other molecular separation methods.The DNA fragments are then visualized and analyzed to identify similarities or differences among the tested clones.IOPRI clones were given a code with the letters MK followed by the serial number of the cloned ortet.The DNA profiles generated from the DNA fingerprinting of ortets can be used as the genetic ID of the clones.An example of the results of DNA analysis can be seen in Figure 4.
In addition to checking the clones in the field, DNA analysis was also carried out to see if there was no change in the DNA pattern from the ortet source used and the clones produced.This is done by comparing the DNA pattern of the ortet source and each culture phase in the laboratory.Until now, DNA analysis of ortet, callus, and embryos has been carried out, and when compared,there is no change in the DNA fragments between the ortet and the culture results, both in the callus and embryo phases.The use of DNA analysis was reported in the publications of [47] and [48].

Field testing, monitoring, and evaluation
The field test was carried out on each clone produced.This test aimed to find out how the performance of clones vegetatively, generatively, and clone production in the field.The tested clones were observed for their vegetative growth, followed by generative observations to determine whether the plants bore mantles or not and to observe other clone abnormalities.The first clone test resulted from the improvement of the method that had been carried out by PPKS through subculture restrictions and culture processes that had been included in a traceable database that had been planted in 2013 in the Riau region to produce clones that were 100% normal.Tests at other plantations showed low abnormality results of <3% in clone testing in the North Sumatra and Jambi areas planted in 2015-2016 (Table 3).In addition to providing low abnormality, clone testing results also provided high clone productivity, uniform plantlets, and high oil content (Table 4).

Fourth decade, the 2020s: Mass multiplication, field testing, and preparation for oil palm clones release and commercialization
The glory of renewal in tissue culture production in the 3rd decade supported by traceable databases and results of clone testing in the field with high productivity and low clone abnormalitiesmotivated PPKS to try to release the clones produced officially.With the release of clone varieties, it was expected that the clones produced by PPKS could be distributed commercially.
Efforts made towards releasing of clone varieties were by conducting mass multiplication or production of PPKS clones, field testing and data collection, making test proposals for the required documents, releasing, and general commercialization of clones (Figure 5 a & 5b).Clone productionwas carried out by re-cloning strategy using ortet sources that had been tested & selected, and had low or no clone abnormality during the test.At the same time, collecting the necessary data was alsocarried out in making proposals for the release of varieties to their commercialization.

Productivity and soma-clonal variation in each decade
The abnormality of oil palm clones in the first decade varied from zero to quite high.Early inthe first decade, testing of the first clones produced from 1987 to 1989 showed very low or almost no abnormality.However, from the 1990s to the end of the second decade, the abnormality increasedfrom 6.16% to 16.60%.This got worse at the beginning of the third decade where the abnormality reached 39.48% with the 2009-2010 planting year.During this era, the clone abnormality found wasthe highest during the decades of PPKS oil palm clone development.However, as previously explained, in the third decade, innovation was carried out with improvements to the PPKS clone production system, so at the end of the third to fourth decades, the results of testing of clones plantedin 2013, 2015 and 2016 in the field showed a very low percentage of clone abnormalities, less than 0-2.69% (  [49] The relationship between abnormality and clone productivity follows a linear graph wherethe higher or the more varied the abnormality of the clones in a plantation, the lower the clone's productivity.Some of the results of clone testing in Figure 6a showed clone testing in various experiments which indicated a relationship between the productivity of fresh fruit bunches (FFB) when stack up with the diversity of abnormality clones represented by the abnormality score, wherethe higher the abnormality score, the greater the variety and severity of the clone abnormalities.Clonetesting was able to produce fresh fruit bunches up to 33.37 tons/ha/year in conditions without clone abnormalities.As the abnormality score increased, clone production decreased and the test results showed that the productivity of fresh fruit bunches was only 14.03 tons/ha/year with the highest abnormality score.When compared between the highest and lowest productivity, it can be said that clone abnormalities can reduce productivity by more than 50% of the clone's ideal potential.Under ideal conditions for clones with low abnormalities, test results showed that clone productivity consistently provided higher yields compared to its comparators or plants from commercial DxP, which was around 20-30% higher.Observations of clone production in one of the PPKS clone testing gardens showed that the FFB of oil palm at four years of age was 4.14 tons/ha/year which then increased with plant age and clone production, and reached its peak at nine years of age with production reaching 32.15 tons/ha/year, which then decreased slightly at the age of 10 years, namely 25.78 tons/ha/year.After 10 years to 14 years of observation, cloned FFB production was sloping (Figure 6b).The following results from testing the PPKS clone compared to the commercial DxP.The data resulted from testing clones with the La Me genetic background, which were compared with the commercial DxP variety DxP La Me.Based on Table 4, the FFB potential of PPKS oil palm clones reached 38 tons/ha/year compared to its DxP of 30 tons/ha/year or the percentage of clones compared to DxP was 126.7% higher.The oil potential character or OY which showed the potential of PPKS clones was 8.4 tons/ha/year compared to DxP, which was only 7 tons/ha/year or 120% higher.Likewise, the character of oil per bunch (O/B) showed that the clone had oil content per bunch was 121.8% higher than the DxP.The vegetative characters, especially the height growth (HI), showed that the height growth of the clones with the DxP tended to be the same, reaching 99.8%.The rachis length character (RL) was quite interesting because the clone's rachis length was shorter than the DxP, namely 4.2 m and 6.1 m respectively, or the clone's rachis length was 69.3% shorter (Table 3).Many plantlets have been produced from these ortet sources.However, 363,133 clone plants were recorded in various clone tests over four decades.Clone distribution has been carried out from Aceh, Sumatra and Kalimantan islands (Figure 7a).Until now, clone testing is still being carried out in several clone testing areas, either independently or in collaboration with state and private companies.

Prospects of oil palm clones
The use of planting material for oil palm clones is very promising in the future.Oil palm clones derived from Elite Tenera clones possess the characteristic of consistently yielding 100% Tenera clones.This means there is no fear of Dura or Pisifera contamination in future clone plantings such as planting material from DxP materials.Another benefit is that the clones produced are genetically identical to the superior ortet source, ensuring true-to-type characteristics.This uniformity translates to higher productivity than the DxP material variety when planted in the same land area, resulting in increased profitability.
The upcoming project is to produce clones that have been tested and selected so that commercialization can be carried out immediately.To support this, plantlet barcoding will be carried out so that it is properly coded and strengthens an integrated database system from the laboratory to the field.This is of course still supported by routine monitoring and evaluation.A flowchart of the plan to support clone commercialization is presented in Figure 7b.
In addition, currently, the majority of PPKS clones are still based on La Me's genetic background.In further developing PPKS clones, there are still many prospects for developing clones with other genetic backgrounds owned by PPKS, like those from the Yangambi, SP540, Dumpy, and OG Hybrid populations.Moreover, mass clones can also be developed by adding new superior secondary a b properties,like compact palm clones, high in vitamin E, high in beta carotene, and resistant to certain diseases, as in Ganoderma.

Conclusion
Some things that can be concluded from the explanation above are the followings: a. IOPRI's clones have been developed since the 1990s and tested in the field, and since 2010 wehave developed clones with high productivity and low abnormalities.b.Culture system improvement for clone production had been carried out and produced positive results.c.Selected clones from field testing will be released and commercialized as superior planting materials.d.The future outlook for the oil palm clone industry is highly promising, with its exceptional productivity and continuous development using diverse genetic backgrounds.

Figure 2 .
Figure 2. The relationship between the length of media exposure and the percentage of clone abnormalities.Source: [25]

Figure 3 .
Figure 3.a) initial version of the database system; b) & c) the latest version of the database system

Figure 4 .
Figure 4.An example of DNA profiles of clones by using SSR_PCR

Figure 5 .
Figure 5. a) Oil palm tissue culture process in the laboratory & clone testing in the field; b) Processflow towards commercialization of oil palm clone varieties.

Figure 7 .
Figure 7. a) Distribution of clone plantings over 4 decades; b) Future plans for oil palm clones commercialization

Table 1 .
The relationship between the number of sub-cultures and the percentage of mantled palms at the embryo stage

Table 2
). a b

Table 2 .
Oil Palm Somaclonal variation in every decade

Table 3 .
Productivity of oil palm clone vs DxP

4. Clone and Field Testing Status During
the 4 decades of development of the PPKS oil palm clones until March 2023, a total of 921 ortets had been planted from various test sources with the following details (Table4):

Table 4 .
Details of the number of ortets having been cultured per decade