Stable Transformant of Phalaenopsis amabilis Somatic Embryo Carrying 35S::AtRKD4 Develops Into Normal Phenotype of Transgenic Plant

ABSTRACT Phalaenopsis amabilis (L.) Blume orchid is an Indonesian national flower. The number of these orchids in their natural habitat is very limited, therefore plant propagation efforts are needed. One of the promising methods is plant propagation by inserting embryo gene AtRKD4 from a model plant Arabidopsis thaliana into the orchid genome to produce many somatic embryos. From previous research, we have obtained 28 plant P. amabilis transformants carrying the AtRKD4 gene, however, it was unknown whether these plants have normal phenotypes and growth similar to their parents. Therefore, descriptions on growth and morphology are needed. This research aimed to evaluate the phenotype of P. amabilis carrying 35S::AtRKD4 the transformants grown in greenhouse. To achieve it, AtRKD4 gene integration stability on transformants genome was analyzed. Morphology and cross-section anatomy structure on transformant and non-transformant plants were described. The stability of AtRKD4 gene integration in the plant genome was confirmed by amplification of the AtRKD4 gene from genomic DNA with Polymerase Chain Reaction (PCR) using a specific primer for AtRKD4 and ACTIN genes as the internal control. The quantitative data from morphology and anatomy measurements were analyzed statistically using ANOVA. The results showed that AtRKD4 was stably integrated into the genome of P. amabilis transformants and all transformant plants showed similar morphology and anatomy characteristics as non-transformant plants. The AtRKD4 embryo gene was stably integrated into the orchid genome and the transformant plants grow normally without significant changes in phenotype.


INTRODUCTION
Orchid is one of the commercial plants in the field of floriculture (Pamarthi et al. 2019) and has high economic value (Palma et al. 2010). One of the important orchids is Phalaenopsis amabilis (L.) Blume, which is commonly called the Moth Orchid, which produces beautiful flowers shaped like a moth. The beauty of P. amabilis flowers has made it called "Puspa Pesona" Copyright: © 2021, J. Tropical Biodiversity Biotechnology (CC BY-SA 4.0) which is used as one of Indonesia's national flowers, along with jasmine and Rafflesia arnoldii (Schuiteman 2010;Semiarti et al. 2018). The uniqueness of this P.amabilis flower is the presence of an antenna-like structure at the end of its labellum, which always appears in the flower of this orchid's hybrid so that P. amabilis is commonly used as a parental species for breeding purposes to produce superior Phalaenopsis hybrid in the world (Semiarti et al. 2011).
Generative/sexual propagation depends on the success of the pollination process. It is ineffective because it takes a long time to produce a new plant (Shekarriz et al. 2014;Mursyanti et al. 2015;Saputro et al. 2018). Somatic embryogenesis is an effective method for orchid propagation. It can produce large numbers of uniform plants in a relatively short time (Feng & Chen 2014;Mose et al. 2017;Yang et al. 2020). The development of methods through genetic transformation could be beneficial for increasing crop production and conservation (Semiarti et al. 2007;Hsing et al. 2016;Zulwanis et al. 2020).
It is well known that plant development is controlled by gene regulation. The RKD4 gene is a member of the RWP-RK protein family in a model plant Arabidopsis thaliana encoding transcription factors for gene expression in early plant development (Waki et al. 2011), promotes zygote extension (Jeong et al. 2011), induces somatic embryogenesis (Zimmerman 1993) and induces cell proliferation and expression of an egg cell (Kőszegi et al. 2011). The insertion of AtRKD4 gene from A. thaliana succeeded in inducing somatic embryo in the Phalaenopsis "Sogo Vivien" orchid hybrid (Mursyanti et al. 2015) and Dendrobium phalaenopsis (Setiari et al. 2018). So far, there has never been comprehensive studies regarding the influence of ecological factors on the growth of transformant plants during the acclimatization period after being transferred from in vitro condition which is strictly controlled to ex vitro condition, in which various environmental factors highly influence it. In fact, the process of transformant plants going through the acclimatization period to be able to grow normally in ex vitro condition is crucial. Therefore, we need to ensure that the transformant plants produced by somatic embryo induction from AtRKD4 expression can grow and develop similarly as non-transformant plants.

Plant Material and Growth Conditions
The plant materials used in this study were ten non-transformant of wild type P. amabilis (Yogyakarta Forms) plants age 8-10 months and 10 plants of P. amabilis transformants, carrying the 35S::AtRKD4 gene, obtained from previous research results of the Orchid Research Team Faculty of Biology UGM. Each plant was planted in pots with a combination of fern and wood charcoal (1: 1), they were maintained in a greenhouse under natural sun lighting environment (28-31˚C) and relative humidity ranges 74-80%.

Detection of AtRKD4 gene in the genome of P. amabilis transformants
Isolation of the whole genome DNA of wild type and transformants P. amabilis was performed by the standard method of plant genome DNA isolation as described by Semiarti et al. (2007) by using Cetyltrimethyl Ammonium Bromide (CTAB). Detection of the existence of AtRKD4 gene in the transformants was performed using Polymerase Chain Reaction (PCR) with KOD FX NEO PCR Kit (Toyobo) and specific primers for AtRKD4 gene and Phalaenopsis actin gene (ACT4) (Yuan et al. 2014) as the internal control (Table 1).
PCR reactions for both genes were performed in 3-step-cycles: an initial denaturation 94°C for 2 min, denaturation at 98°C for 10 seconds, annealing at 58 °C for AtRKD4 and 51°C for ACT4 for 30 seconds, and extension at 68˚C for 6 sec seconds using a Thermal Cycler (T100, Biorad). PCR steps were performed in 35 cycles. The amplified DNA fragments were checked with 0.8% agarose gel (Agarose Type II, SIGMA).

Morphological Analysis
Plant growth was observed every week for eight weeks to get detailed similarities and differences in growth between P. amabilis transformant and non-transformant plants, ten plants each for both non-transformant and transformant plants. Plant morphological data covering the total number of leaves, leaf length, leaf width, as well as the total number of roots, root length and root diameter were observed and measured weekly. The phenotypic analysis includes the shape and color of leaves, roots, and survival ability. The observations were documented using a digital camera (EOS M100, Canon), color characteristic was determined using RHS color chart (The Royal Horticultural Society, London).

Anatomical Structure Analysis of Roots and Leaves
Anatomical preparations of roots and leaves cross-section were conducted using the paraffin embedding method according to Sutikno (2016) and stained by 1% safranin in 70% alcohol, the sample was fixed with FAA solution (Formalin: glacial acetic acid: 70% alcohol = 5: 5: 90). Observations of the anatomy slides were performed by using a light microscope (CX22LED, Olympus) and documented using a digital microscope camera (Optilab Advance V2, Miconos).

Statistical analysis
Quantitative data is processed using Microsoft Excel 2013 applications and Raster 3 image, then statistically analyzed using IBM SPSS Statistics 26.0 to find out whether there are growth and phenotype differences. Analysis of Variance (ANOVA) was conducted to test the significance difference of the growth by both groups with significance level 0.05. Qualitative data was descriptively analyzed.

P. amabilis transformants and non-transformants growth
Growth of P. amabilis transformant and non-transformant plants were observed for 8 weeks. The phenotypes of P. amabilis transformant and nontransformant plants showed very similar growth patterns (Figures 1 and 2). Observation was conducted as a first step to determine morphological characteristics of the two groups.
During acclimatization, P. amabilis non-transformant and transformant orchids at week 0 ( Figures 1A and 2A week one ( Figures 1B and 2B) the leaves began to undergo chlorosis at the tips. P. amabilis non-transformant orchids began to adapt in ex vitro environment at week 4 ( Figure 1D), which marked by new root and leaf growth after 5-week acclimatization ( Figure 1E). Whereas, P. amabilis (T) at week 2 ( Figure 2C) have shown the ability to adapt in ex vitro environment characterized by the growth of new roots and leaves on week 4 ( Figure 2E). Mirani et al. (2017) reported a similar phenomenon in Dendrobium nobile acclimatization. Maintenance of humidity and temperature around the plantlets during the initial days of acclimatization is of utmost importance. During in vitro culture, plantlets were grown in high humidity conditions in jars. Meanwhile, in greenhouse or field has much lower humidity than in vitro condition. Therefore, rapid changes in environmental conditions, especially in terms of temperature and humidity, could led to wilting and chlorosis. P. amabilis non-transformant and transformant have an obovate leaf shape, where the widest part at the tip of the leaf blade. The shape of the leaf tip is retuse and the leaf edge is integer (Figures 1 and 2).  A significance level above 0.05 indicates no difference in growth between P. amabilis non-transformant and transformant plants. As shown in Table 2, P. amabilis non-transformant and transformant plants had  Semiarti et al. (2018) who compared the vegetative growth between Phalaenopsis "Sogo Vivien" carrying 35S::Gal4::AtRKD4::GR and the non-transformant plants that showed no change in plant phenotype and growth condition.

Anatomic structure of roots and leaves of P. amabilis transformants and non-transformants
Cross-section of root anatomical structure ( Figure 3) and leaf anatomic structure (Figure 4) shows high similarities in the shape and size of the leaf and root cells between transformant and non-transformant plants. There were no significant differences between transformant and non-transformant plants on the anatomic structure of roots and leaves.  The cross-section of roots of P.amabilis orchid transformant and nontransformant showed a very similar anatomical structure consisting of Rhizome, Cortex and Stele (Figure 3). Table 3 shows that the size of the length and width of these cells did not show a significant difference of Rhizome, Cortex and Stele with ANOVA significance of 5%. The anatomical structure of the leaves (Figure 4) also did not show significant differences in both the structure and size of cells Epidermis, vascular bundle (Xylem and Phloem), and parenchyma (Table 4). This phenomenon indicated that the activity of the AtRKD4 gene in somatic embryo initiation did not affect on the growth and further development of the orchid somatic embryos, only at an early stage for the initiation of somatic cells into embryos. The AtRKD4 gene, therefore, has no impact on the activity of growth genes to continue the growth and development of embryos into complete plants. This fact is highly expected by genetic engineers for P. amabilis orchid's mass propagation as a conservation effort as well as for producing large number of plants for agribusiness purposes.

Detection of the integration of AtRKD4 gene in the genome of P. amabilis transformant
Positive amplification results of genomic DNA with specific primers for AtRKD4 gene and primers for housekeeping gene ACTIN as an internal control for PCR reaction showed that a 114 bp of ACTIN fragment was amplified from the genome of both P. amabilis non-transformant and transformant, whereas 198 bp of AtRKD4 fragment was amplified only from the genome of P. amabilis transformants ( Figure 5). This data indicated that the AtRKD4 gene is stably integrated in the genome of P. amabilis transformants while the transformant plants grow and develop similarly as non-transformant plants. Waki et al. (2011) reported that in the Arabidopsis thaliana plant, the AtRKD4 gene produced the RKD4 protein triggers gene expression and pattern formation in early embryogenesis. This is also supported by research data on somatic embryogenesis induction on Phalaenopsis 'Sogo Vivien' from Mursyanti et al. (2016) and research by Setiari et al. (2018) in somatic embryogenesis of Dendrobium phalaenopsis. This is also in accordance with Mose et al. (2020), who succeeded in inducing somatic embryogenesis by treating growth regulators thidiazuron and NAA from protocorms and P. amabilis leaf pieces as explants.  CONCLUSION P. amabilis transformant plants remain stable on carrying AtRKD4 gene and grew normally as the same as non-transformant plant. It has great potential to be used as an explant donor for the propagation of P. amabilis plants for various purposes, including production of seedlings for both conservation and agribusiness purposes. Overall, morphological data, anatomical data, and molecular data support the use of the AtRKD4 gene for orchid propagation for the conservation of P.amabilis orchids whose population is decreasing in nature, as well as orchid production for economic purposes.