Improvement of Selected Induction Culture Media on Callus Induction in Anther Culture of Anthurium and a Histological Study on its Callus Formation

Improvement of selected induction culture media on callus induction in anther culture of anthurium and a histological study on its callus formation were studied at the tissue culture laboratory of the Indonesian Ornamental Crops Research Institute from February to October 2008. The objectives of the study were to optimize selected media for callus formation, reveal cell origin of callus derived from anther culture and shoot formation process. Selected media improved in the study were 1) MMS-TBN containing 0,5 mg/l TDZ, 1,0 mg/l BAP and 0,01 mg/l NAA (Winarto medium, WM) and 2) MMS III supplemented with 1,5 mg/l TDZ, 0,75 mg/l BAP and 0,02 mg/l NAA (Winarto and Rachmawati medium, WRM). Improvement treatments were carried out by omission and application of 2,4-D in 0.5 mg/l and reduction of medium strength of full, half, quarter, one eighth, one sixteenth, and zero strength. A factorial experiment was arranged using a randomized complete block design with four replications. Results of this study indicated that the highest callus induction was clearly established in WRM. The medium stimulated potential growth of anther (PGA) up to 81% with 49% of percentage of anther regeneration (PAR) and 2.7 number of callus formed per replication (NCF). Significant improvement in callus formation was also recorded by reduction of medium strength of WRM to one eighth compared to others. The reduction induced PGA up to 58% with 29% of PAR and 1.8 NCF. From histological studies it was well recognized that regenerated callus on half anthers cultured was originated from middle layer cells of anther wall. The morphogenic response of anther wall cells caused primarily on no androgenesis effect in microspore cells.


INTRODUCTION
Application of anther culture and/or microspore culture in ornamental crops till now is still limited. The technique was reported in several plants such as on lily (van den Bulk et al., 1992;Han et al., 1997), tulip (Tanaka andIto, 1981 &1982;van den Bulk et al., 1994), sunflower (Saji & Sujatha 1998), petunia (Mohan-Jain & Bhalla-Sharin 1996), Camelia japonica (Pedroso & Pais 1996). While in Araceae, especially in anthurium, its application was very limited. In Araceae, double haploid plant production was tried in Spatiphyllum via ovule culture (Eeckhaut et al., 2001), but number of double haploid plant produced was very low. induce callus and its regeneration (Rachmawati 2005;Winarto & Rachmawati 2007). In a comparative study it was also found that MMS supplemented with 0.5 mg/l TDZ, 1.0 mg/l BAP and 0.01 mg/l NAA (Winarto medium, WM) was another potential medium in anther culture of anthurium (Winarto et al., 2009).
Two different calluses derived from anther that a part of them often showed different colors in their performance (green and yellow in Tropical and Amigo cultivars; red-purple and yellow in Carnaval cultivar) and growth types (slow and fast) interested to be studied in detail via their histology. From the histological study it was expected that callus origin could be clearly known.
In the first time it was hypothesized that the slow growth callus was initiated from microspore cells and the faster one was derived from anther wall and/or connective tissue cells. Important role of the study in in vitro cultures was generally carried out to support and strengthen research results. The study was applied in anthers of Vitis rupcstris (Altamura et al., 1992), in androgenesis of rice (Oryza sativa L.) (Nakano & Maeda, 1989;Mandal & Gupta, 1996), in callogenesis and organogenesis of Curcuma zedoaria Roscoe (Mello et al., 2001), in petiole derived callus of Amorphophallus rivieri Durieu (Hu et al., 2005). Improvement of induction culture medium in callus initiation and formation via application of 2,4-d and/or increasing its concentration was actually contributed in increasing morphogenic response of the anther walls and connective tissues to divide actively and produce callus as stated by Rodrigues et al., (2004) in soybean anther culture. Enhancing callus initiation due to 2,4-D treatment was also recorded by Thangene et al., (1994) in anther culture of sunflower, Arzate-Fernandez et al., (1997) in lily, Oggema et al., (2007) in sweet potato, (Kumar & Kanwar 2007) in Gerbera jamesonii.
Strengthening medium capacity in callus induction was also recorded successfully by reduction of the medium strength to be half, quarter, or others as reported by Hoque and Arima (2002) in callus induction of water chestnut (Trapa Japonica Flerov), Chen et al., (2005) in Bupleurum kaoi Liu, Jabeen et al., (2006) in Aconitum heterophyllum, (Wang & Bao 2007) in Viola wittrockiana.
Furthermore application of 2,4-D and reduction of medium strength in anthurium anther selected medium expected could improve medium capacity in stimulating high response of the anthers cultured for producing callus.
The objectives of the present investigation were to improve selected induction culture medium in anther culture of anthurium by 2,4-D application and reduction its strength on callus formation and to reveal from which cell and/or tissue actually the regenerated calluses on anther culture derived. watering them. Spadixes with 50% of its pistil in receptive condition were harvested from the plants.

Preparation
2 cmIn the first step, spadixes were placed under tap water for 30-60 minutes to reduce high contamination, followed by immersing in pesticide solution (1% of benomil and bactomycin) for 30 minutes and then rinsed by distillated water for 5-6 times with 5 minutes each.
After first step sterilization, the spadixes were brought into laminar air flow cabinet for next sterilization.
Explants were soaked in 2% of sodium hypochlorite (NaOCl) plus 5 drops of Tween 20 for 5 minutes, 1% of NaOCl added by 5 drops of Tween 20 for 10 minutes, Pistachia vera (Ahmad 1993). Explant wounding induced stress and causes an increase in phenylalanine ammonia lyase (PAL) activity. High activity of PAL enhanced phenylpropanoid production that lead to explant browning (Tabiyeh et al., 2005). Another study reveals that the explant slicing led to cell damage and followed by sub-cellular material mixing from cytoplasm and vacuole (Laukkanen et al., 1999). Just after the occurence, phenolic compounds were oxidized by polyphenol oxidase enzyme to produce quinone compounds and polimer complex due to the existence of O 2 (Marshall et al., 2000). The compounds were toxic and caused browning and death of explant (Stom et al., 2006;Ozygit et al., 2007).  Oggema et al., (2007) in sweet potato, however increasing the 2,4-D concentration let to reduction of callus number initiated. Raghavan (2004) also found existence of cell expansion and division activity during callus formation of cotyledone of Arabidopsis due to 2,4-D application. In Bupleurum kaoi the highest callus weight was observed on half-strength MS medium containing lower concentration (0.1 to 0.2 mg/l-1) of 2,4-D and increasing of its concentration did not further improve callus proliferation (Chen et al., 2005). The result gave evident that application of 2,4-D in anther cultured was still questionable. Improvement of anthurium anther selected media via medium strength reduction. The study also revealed that though the experiment result was not as high as the previous study, but the WRM was still the most appropriate induction culture medium on callus induction compared to W M-D ( Table 2)  formation in anther culture of anthurium as recorded by Hoque and Arima (2002) in Water Chestnut (Trapa Japonica Flerov), Chen et al., (2007)  lawrenceanum 'Tradition') were easily induced from seedderived protocorms on a 1/2 strength Murashige-Skoog medium plus 1-10 mg l "1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1-1 mg l "1 1-phenyl-3-(1.2.3-thiadiazol-5yl)urea (TDZ) (Lin et al., 2000).

The histological study of callus formation.
Surprisingly It could also be reported that high morphogenic anther wall cells were determined from anther wall cells  located between connective tissue and tapetum cells (indicated by yellow arrows in Figure 1 and red circles in figure 3A-C). The active cells producing callus were derived from middle layer cells. In anther culture of rice, callus masses was originally regenerated from connective tissue (Maeda et al., 1978), while in anther culture of Vitis rupcstris, caullogenesis was regenerated from all anther tissue except endothecium (Altamura et al., 1992). In addition from four anther sacs containing microspore cells that were divided into two main parts (contain 2 sacs each) it was only one of main part that indicated high morphogenic response, produced callus and grew faster than another main part ( Figure 3C-E).
Growth direction was indicated by white arrows ( Figure   3D-F).
From the study it was also well recognized that during callus formation microspore cells did not grow . Histological studies of callus formation derived from anther till shoot initiation. A-longitudinal section of anther in initial culture, B-cross section of anther in initial culture, C-D -initial morphogenic response of anther 20 -35 days after culture initiation, F-G -regenerated and developed callus derived from anther 2.0-3.0 months after culture initiation, h-i-initial apical meristem developing 4.0-4.5 months after culture initiation, J-initial shoot developing 4.5-6.0 months after culture initiation. K-Ldeveloped shoots more than 6 months after culture initiation. Red arrows = connective tissue/cells, blue arrows = high morphogenic anther wall cells, green arrows = microspore cells, amd -apical meristem developing, am -apical meristem, vcd -vascular cells developing, vc -vascular cells, ylp -young leaf primordia, yl -young leaf. Blue bars = 0.11 mm, green bars = 0.28 mm, red bars = 0.001 mm