F1 Hybrids in oil palm

F1 Hybrid production in oil palm (Elaeis guineensis) is a reality. F1 Hybrids have the potential to provide massive increases in yield as demonstrated in a range of crops. Verdant has the intellectual property and “know-how” to produce haploids and double them to give doubled haploids. Doubled haploids are fertile and 100% homozygous (genetically pure lines), making them ideal as parents for F1 Hybrids. There has been no genetic modification. Verdant now has a collection of Dura and Pisifera doubled haploids that span the genetic diversity of oil palm from which parents can be selected based on genetic contrasts (using Diversity Array Technology). The first F1 Hybrid crosses (Dura x Pisifera) were made in 2019 and field progeny trial testing started in 2021. Performance in the field is being monitored by a range of physiological tests, but ultimately on palm product yield per hectare plus commercially significant secondary traits, such as disease tolerance, will determine selection. The intention is to start commercial F1 Hybrid seed production in 2029. A phenomics nursery has been set up to correlate seedling physiological traits with yield with the aim of developing early indicators for yield potential and to screen for variation between crosses for 1) Ganoderma tolerance, 2) nutrient uptake, and 3) drought tolerance. Genotyping is also being deployed to identify and select heterotic combinations that result in hybrid vigour.


F1 Hybrids in oil palm are a reality
Verdant has produced the first F1 Hybrids in oil palm.The first 31 F1 Hybrid progenies were field planted in 2021 and a further 42 were field planted in 2022.There are currently 161 F1 Hybrid progenies in the nursery (for planting in multi-locational trials) and more than 100,000 seeds of 300 F1 Hybrid progenies are being processed for nursery and field trial testing.There is a crossing programme to continue production of new/distinct F1 Hybrid crosses in 2023 and subsequent years.Nursery and field performance data are being gathered and yield recording will start in 2023.Verdant expects to begin commercial seed production of F1 Hybrids in 2029.
The F1 Hybrids are Tenera, produced from genetically pure Dura (female) and Pisifera (male) doubled haploid parental lines.The parental lines are being developed from a wide range of oil palm germplasm for which Verdant has IP.The doubled haploids are produced from chromosome doubling 1308 (2024) 012043 IOP Publishing doi:10.1088/1755-1315/1308/1/012043 2 of oil palm haploids.There is potential to produce over 7,000 distinct F1 Hybrid crosses from the parents developed to date.

Background to Verdant Bioscience
Verdant Bioscience was established in 2013 with an aim to develop high yielding, high quality planting material in oil palm, especially through F1 Hybrid production.Verdant has the IP and facilities to produce haploids, doubled haploids and F1 Hybrids in oil palm.As demonstrated in a range of other crops F1 Hybrids provide a proven strategy for oil palm to achieve its physiological oil yield potential of 18.5 tonnes of oil per hectare [1,2].Increasing oil palm's yield per hectare is probably the only real solution to meet the world's increasing need for vegetable oil without further loss of biodiversity because of new oil palm plantings.Verdant's F1 Hybrid approach fuses traditional breeding strategies with the latest methods in biotechnology.
The Verdant Plantation Science Centre is based at Timbang Deli estate, North Sumatra, Indonesia.The centre has laboratories in Cell Biology, Plant and Soil Genomics, Tissue Culture, Pollen, Plant and Soil Nutrition, Bunch and Oil, Agronomy and Crop Protection.These laboratories are allied and serve the Breeding and Seed Production departments.There are also extensive nurseries, seed gardens and field trials (trial sites are located at various locations across Indonesia).The results and analysis of objective breeding trials underpins Verdant's commercial seed production.
Verdant aims to capitalise on hybrid vigour (heterosis) and emulate the spectacular successes achieved in F1 Hybrids in other crops, notably maize where breeders achieved a 600% increase in yields after the introduction of F1 Hybrids in early 20th century.

Haploids and doubled haploids in plants
Haploids plants have the gametic number of chromosomes (n) as opposed to the normal sporophytic chromosome number (2n).In oil palm haploids have 16 chromosomes (n=16) whereas normal oil palm has 32 (2n=32).Haploids (Hs) are derived from gametic cells, i.e., cells of the microspore (that give rise to pollen), and on the female side cells of the megaspore (that give rise to the ovule).Haploid plants tend to have a similar morphology to normal sporophytes but smaller and sterile.Apart from horticultural interest in novel miniature plants haploids have little intrinsic value.Their value is realised when converted to doubled haploids (DHs) by duplicating the haploid chromosome complement (n -2n), doubling restores fertility and the DHs are completely homozygous (genetically pure).
Haploids occur naturally, but at very low frequencies.Haploids can also be induced in large numbers in a wide range of plant species by applying specialized biotechnologies.Doubled haploids have immense value in plant breeding because: 1.They are 100% homozygous.2. They allow accelerated breeding (reduce the time from the initial cross to cultivar registration), for inbreeding crops the end cultivar may be a DH, for out-crossed crops the DHs may be used as parental lines of cultivars.3. Doubled haploidy can be applied at any generation in a breeding programme.4. DH lines meet official requirements for cultivar status in being distinct, uniform and stable.5. Wide application -protocols are available for over 200 plant species.
DH oil palm parents will produce genetically uniform F1 Hybrid crosses.Therefore, an oil palm seed customer will no-longer receive a consignment of undefined commercial crosses but individual proven genetically uniform crosses.Since the F1 Hybrid crosses are genetically uniform the provenance of commercial material can easily be demonstrated with genetic markers.
Haploids and doubled haploids also provide valuable tools in research and development, e.g., in genetic mapping, transformation and mutation.A brief history is given below.

Brief history and the impact of haploids and doubled haploidy in plant breeding
There have been many reviews written on the evolution and success of doubled haploidy in plant breeding [3,5].The history began with the observation of naturally occurring haploids.Although rare in nature and considered as a novelty, enterprising plant breeders exploited these in producing the first doubled haploid cultivars.However, the low and variable frequency of natural haploids ruled them out for most plant breeding programmes.A breakthrough was the development of high-throughput anther and microspore culture techniques, initially in Datura, but developed later in a wide range of crop species.Here we list some of the milestones: • 1974 First International symposium on haploids in higher plants, Geulph, Canada [9].
• 1979 'Mingo' barley licensed as a cultivar, it is the first DH cultivar produced by induced haploidy and doubled haploidy.This is a landmark achievement as cv.'Mingo' wasregistered in record time (5 years after the initial cross, halving the breeding time) and establishes doubled haploidy as an accelerated breeding technique, [10].• 2001-2006, a five-year EU funded COST Action 851 on: "Gametic cells and molecular breeding for crop improvement", involving collaboration among laboratories and research institutes in 26 European countries.An outcome was the identification of four standard protocols (barley, oilseed rape, tobacco and wheat) which could be tested and adapted for other species, [11].
Doubled haploid breeding provides an accelerated method for cultivar production, doubled haploidy can be applied at any generation in the breeding cycle.In in-breeding species (e.g., barley, rice, wheat, etc.) the DHs can become cultivars directly as they meet regulatory standards for cultivar registration, such as uniformity (as well as being distinct and stable).For out-breeding species (e.g., oil palm, maize and rye) the DHs can be used as parental lines to produce F1 Hybrid cultivars.Doubled haploidy has become a standard breeding method in many crops and many companies that did not convert to doubled haploidy became uncompetitive and no longer exist.Another indicator of the importance of haploidy in plant breeding is the number of related patents, there are over 30 granted patents in haploidy in a 20year period from 1986 [12].

Development of F1 Hybrids in oil palm
The history of doubled haploidy goes back to the 1920s, since then many hundreds of cultivars have been produced in a wide range of crop species, this begs the question -why has it taken so long for oil palm?The answer is largely due to the fact that oil palm has remained recalcitrant to the favoured in vitro techniques of anther and microspore culture.The Verdant approach is founded on exploiting naturally occurring haploids.These occur at a frequency of 1 in 100,000 seed, which in most species would be prohibitively low for breeding purposes.However, oil palm produces seeds in abundance, about 1,000 seeds per bunch in mature palms.In commercial seed production poorly germinated ("offtype") seed are routinely screened out, and the frequency of haploids in "off-type" seed is circa 1 in 100.The haploids in "off-type" seed can be detected easily by flow cytometry.The conversion of haploid germinated seeds and seedlings to doubled haploids can be done by chromosome doubling treatments.Success in producing the first haploid plants in oil palm (over 1,000) and one doubled haploid was reported by Nelson et al. in 2009 [13] and reviewed by Dunwell et al. [14].The selection techniques and methods to increase the haploid frequency have been developed further.The method is based on the natural occurrence of haploids, and Verdant has not used genetic modification in any areas of its breeding programmes including its F1 Hybrid strategy.

Verdant's oil palm germplasm
Verdant has an extensive germplasm collection which covers material from Nigeria, Cameroon, Ivory Coast and Zaire, as well as breeding populations and commercial material.These have been genetically fingerprinted using Diversity Array Technology (DArT) [15].The genetic diversity between these groups is displayed in Figure 1.(for details see Nur et al [16]. Genotypes in the various groups (Figure 1) have been targeted to produce Hs/DHs to provide a wide range of parents for F1 Hybrid production.At this stage accurate predictions on which combination are best cannot be made but are being tested.Various strategies have therefore been developed to maximise the chances of success.These include: • Commercial x Commercial crosses which will provide commercially acceptable progenies, but yield increases are expected to be modest.• Commercial x Non-commercial crosses in which improvements that break current commercial yields are expected.• Non-commercial x Non-commercial, these crosses are more speculative, but likely to produce some exceptional progenies.• Maximising the genetic difference between parents, the greater the diversity between parents the greater the likelihood of capturing heterosis.Examples are given in figure 2.  Verdant is developing haploids and doubled haploids from a wide range of oil palm germplasm.It takes five years before a doubled haploid palm can be selfed (in the case of Duras) for seed palm multiplication.For the female sterile Pisifera DHs these can be cloned via tissue culture.Hybrids have been planted in the field, the first batch planted in 2021 includes 31 progenies and the second batch planted in 2022 includes 42 progenies.In the pipeline there are 161 F1 Hybrid progenies in the nursery, these are intended for multi-locational trials to assess performance in different environments.Seeds for 300 F1 Hybrid crosses made in 2022 are at the germination stage.Nursery and field data are recorded continually, and the first yield data will be available in 2028.Commercial seed production of F1 Hybrids is expected in 2029.

Next steps 5.1. Seed gardens and pollen gardens
Verdant aims to provide F1 Hybrids to the market as soon as robust field-testing data are available, with a target date for the release of the first cultivars in 2029.In preparation for commercial production parental lines are being multiplied to be planted in seed (Dura) and pollen (Pisifera) gardens.The Dura DHs are relatively easy to propagate as this can be done by selfing using standard procedures.For sterile Pisifera DHs cloning is done using tissue culture techniques.

Finding heterotic loci, genomic prediction applications
A major breeding objective is to determine the genetic controls of heterosis (hybrid vigour).Verdant is conducting a range of crosses between diverse parental lines which have been genotyped using irecti n m cro s ween s.

Figure 1 .
Figure 1.Genetic diversity of Verdant germplasm displayed as clusters in a 3D PCoA (for details see Nur et al [16].

Figure 2 .
Figure 2. Direction of some crosses made between different PCoA groups.
1921 Bergner reports natural haploid plants in Jimson weed, quickly followed by similar findings of haploids in other species [3].• 1925 'Marglobe' tomato, the first doubled haploid cultivar is bred from a spontaneous haploid, released as a cultivar in the US [4].• 1952 Chase introduces haploids and doubled haploids in maize breeding [5].• 1963 Kimber and Riley assess and review the exploitation of haploids and doubled haploids in plant breeding [6].• 1964 Guha and Maheshwari generate haploids in large numbers via anther culture in Datura [7].• 1970 Kasha and Kao report a method to produce high numbers of haploids in barley.[8].

Table 1 .
Characteristics of groupings in the PCoA.