Recent Developments in F1 Hybrid Project

London Sumatra (Lonsum) has continuously pursued the F1 hybrid project to produce the genetic and phenotypic uniformity of palms that highlight the exceptional qualities of the parents. Selection of haploid palms that have been duplicated by tissue culture techniques to become doubled haploid is an efficient method for producing highly homozygous palms. About 206 dura haploids and 47 haploid pisifera from different populations were obtained by abnormal seed and seedling screening. During this time, one of four dura-doubled haploids that produce normal flowers have proliferated. Due to the unavailability of doubled haploid pisifera, the potential of dura doubled haploid as a parental candidate was tested by crossing it with highly homozygous diploid pisifera (>80%). The offspring are referred to as semi-hybrids and are currently in an immature state. Going forward, the F1 hybrid project will cross doubled haploid dura and pisifera while continuing to monitor the potential of semi-hybrids.


Introduction
As part of the oil palm industry, Lonsum continues to develop strategies to improve the quality and quantity of oil palm.One of the approaches is the production of the F1 hybrid.An F1 hybrid is a term used to describe first-generation seeds/plants that result from the successful cross-pollination of a genetically uniform plant variety with another specific genetically uniform variety.There are many examples of F1 hybrid crops showing benefits today, for example in wheat [1], sunflower [2] or rice [3].There are also different approaches to producing F1 hybrids, ranging from controlling pollination (e.g.hand pollination) to crossing the inbred parent lines of two specific plant varieties to crossing two dihaploid parents.The key to the F1 hybrid is the focus on the two parental lines, which share a high degree of homozygosity in their genetic material.
An F1 hybrid is the result of crossing two pure lines to achieve the desired result that appears to offer many benefits.Through this approach, it is not only possible to highlight the outstanding traits of the parent plants, but in most cases these traits have also been enhanced and new desirable traits added to the resulting hybrid plants.In addition to traits such as good vigor, they produce higher yields, higher seedling survival rates, have a more uniform appearance, and are resistant to disease and insects [4].
Despite the introduction of F1 hybrid varieties with excellent yield increases in a variety of crops, oil palm F1 hybrids have yet to be introduced due to the difficulty of producing homozygous elite parents.This is due to the fact that inbreeding requires a long oil palm generation time (six to eight selfpollinating generations to produce sufficient inbred lines (nearly homozygous) with a current minimum generation time of four to five years).However, due to heterosis (hybrid vigor), genetic uniformity allowing for greater optimization of agronomic practices, and the opportunity to exploit genotypeenvironment (G x E) interactions, oil palm F1 hybrids are expected to significantly increase yield per hectare [5].
Lonsum started the F1 hybrid project by screening the haploid seeds or seedlings, the first step in producing doubled haploid palms, which in turn provide parent lines for F1 hybrid production.Over the years, Lonsum conducted a large-scale morphological off-type screening on oil palm seeds and seedlings produced by Bah Lias Research Station (BLRS) in Indonesia.These phenotypically abnormal seeds and seedlings are then subjected to ploidy analysis to confirm their haploid chromosome [6].Subsequently, the haploid plants are subjected to a tissue culture method to produce fully homozygous true breeding parent lines, so-called doubled haploids, which arise spontaneously during the first mitoses of embryogenesis [7].The first haploid was found in 2003, while the first doubled haploid was confirmed in 2010.On the other hand, since pisifera doubled haploids are not yet available, Lonsum chose another alternative way to predict the results of the F1 hybrid project by crossing the dura doubled haploid with highly homozygous pisiferas from the conventional breeding method.This report aims to provide the latest update on the F1 hybrid project and potentially contribute to the oil palm breeding program.

Haploid Screening
Haploid describes an organism that contains a single set of chromosomes in a cell.The haploid oil palm consists of 16 chromosomes, which is half the set of chromosomes present in the normal oil palm (known as diploid).There are two screening methods with specific criteria for each method as described in the figures below.

Flow Cytometry Analysis
The suspected haploids were monitored and maintained until ready for ploidy analysis using the flow cytometry method.Leaf samples were cut into small pieces in labeled plastic petri dishes on ice gel in nuclear isolation buffer and then incubated in the laboratory refrigerator for 30 minutes.The incubated samples were filtered through 30 μm cell filters.The samples were then loaded into a flow cytometer (Partec CyFlow PA) to determine their ploidy level.

Seedlings Growing, Haploid Selection and Haploid Cloning
Haploid palms are nurtured through several stages before they are ready for cloning by the tissue culture laboratory: pre-nursery, main nursery, and field nursery.Haploid selection is performed to evaluate which haploids could be cloned by a tissue culture technique with the intention of propagating the haploids or spontaneously doubling the chromosome to become doubled haploids (DH).When the stem diameter reaches 20 cm, the haploids can be cloned by the tissue culture laboratory.Leaf samples were taken from ramets for ploidy analysis to ensure that the clones had already formed doubled haploid (DH) palms.

Homozygosity Analysis
Homozygosity analysis is the method of determining the percentage of gene uniformity of the organism.Ramets were selected based on amplification of 43 Simple Sequence Repeat (SSR) markers.For the next steps (field planting, flower counting, self-pollination and progeny testing) only DH clones with a homozygosity percentage of 95% would be selected.

Flowering Census
Planted DH palms that produced flowers were subjected to the flower count, which was performed to identify normal and abnormal female flowers of oil palm clones.

Multiplying Doubled Haploids and Parental testing
Doubled haploids of Deli dura that produced normal flowers were propagated by self-pollination and were to be crossed with DH pisifera to produce an F1 hybrid.However, due to the unavailability of DH pisifera, DH dura was crossed with four different highly homozygous (HHD) pisiferas with >80% homozygosity as male parents to realize its potential.The offspring of these crosses were called semihybrids.

Morphology and Physiology Characterization
Multiplied DH palms were characterized by their morphological and physiological performances, while semi-hybrids were characterized only by their physiological performances to identify similarity or dissimilarity to diploid palms (compared to standard crossbreeds).

Results and Discussion
The probability of getting haploids was higher in seedling screening for both dura and pisifera than in seed screening.With the abnormal seed screening method, the probability of getting haploid was 0.034% while screening for abnormal seedlings was 1.45%.So far, Lonsum has collected 206 dura haploids and 47 pisifera haploids.In addition, the proportion of haploids from pure populations is also lower than in mixed populations (Figure 3), as Lonsum has put more effort into finding only haploids from pure populations in recent years.Meanwhile, it is unfortunate that with so much effort to find the haploids, the haploid death rate is also very high.These are caused by several factors such as haploids being genetically weak materials, being more susceptible to pests and diseases (infection by saprophytic fungi mainly in the rainy season, Oryctes sp. or Adoretus sp. or after crown sampling prior to cloning), being physically abnormal (smaller than diploid palms), and having more specific nutritional needs.
Many reports mentioned that haploids have unusual characteristics, and morphological studies showed that the haploid plant was shorter, grew more slowly, developed smaller leaves, flowers and stomata, and was often small and weak in appearance [8] and [9].In addition, significant problems are encountered in the development and growth of haploids in vitro, which can make tissue maintenance and regeneration difficult and can even lead to tissue degradation.Poor performance of haploid tissue may be related to increased expression of recessive, lethal, or sub-lethal genes compared to heterozygous material.In the case of haploids, the lack of a homologous set of chromosomes also means that the plants are sterile [10].Another challenge when working with haploids is the difficulty of creating doubled haploids with 100% homozygosity through the cloning process.It is interesting to see that only about 14% of them produced 100% homozygous DHs (Figure 4).The level of homozygosity, which was below 95%, could be due to genetic and epigenetic changes induced by the tissue culture method during doubled haploid production, a phenomenon collectively called soma-clonal variation.This can lead to a genetic mutation that changes the variation in genes such as alleles.This phenomenon was previously studied in pure F1 hybrids and rice polyploids [11], but is poorly understood in the oil palm.As previously reported, the achievement of fully homozygous plants is probably due to morphogenetic processes that do not allow the plants to regenerate due to their unfavourable culture conditions [12].And, as is usual with tissue culture, the abnormality of the flowers became another problem with this approach, as only 15% of the DHs produced normal flowers (Figure 5).This is likely due to the direct relationship between oil palm DNA methylation and the determinism of the enveloped soma-clonal variant, as previously reported [13].

Figure 5. Percentage of flower types produced by DHs
In addition, Lonsum has also multiplied the DH dura and continued to characterize its morphology and physiological performances.The offspring were planted and cared for using standard (nursery and field) procedures.Interestingly, it was shown that there were no significant differences between the progeny of DH dura (self-pollinated) and standard diploid dura crosses (table 1).Theoretically, it was predicted that the offspring from self-pollination, which have a higher level of homozygosity, would perform poorly in terms of disease resistance, growth and yield compared to standard diploid crosses, as observed in other plants due to inbreeding effects, as previously reported [4].However, since these were only annual palms, more data from subsequent years is needed to describe the actual performance of these DH palms.In theory, the D x P F1 hybrid is the result of crossing two fully homozygous parental lines, dura and pisifera.However, since DH pisifera was not available, Lonsum decided to use HHD pisifera instead to assess the potential of the offspring, referred to as semi-hybrids.The physiological performances of annual palms were compared to standard crosses, with no significant differences being found in most of the parameters performed (table 2).As other reports have stated that hybrids are expected to outperform their homozygous parents in areas such as superiority vigor [4], records should be kept for the next few years to assess their potential morphological and physiological traits.

Conclusion
It is interesting to note that screening of abnormal seeds and seedlings emerged as an effective method for collecting haploid oil palms, while tissue culture cloning was the method for generating doubled haploids as parental lines.On the other hand, annual propagated DH palms and semi-hybrids show no differences in physiological traits from standard crosses and should be monitored and recorded for the next few years.

Figure 1 .
Figure 1.Abnormal seed (a) and seedling (b) criteria for finding the suspected haploids

Figure 2 .Figure 3 .
Figure 2. Haploids (A) and doubled haploids (B) of Deli dura planted in the field trial

Figure 4 .
Figure 4. Percentage of DHs generated by cloning tissue cultures with their homozygosity

6 Figure 6 .
Figure 6.The offspring of DH Deli dura planted in the nursery (A) and in the field trial (B)

Figure 7 .
Figure 7.The semi hybrid from crossing DH Deli dura with highly homozygous pisifera

Table 1 .
Comparison of morphology and physiological characterization of standard crossbreeds (SC) and multiplied doubled haploids (DH)