EFFECT OF THE COMBINATION OF THE GROWTH REGULATORS AND PUTRESCINE ON THE SOMATIC EMBRYOGENESIS OF WHEAT ( Triticum aestivum L.) ON SOME TYPES OF EXPLANTS

. Plant breeding programs need to be carried out in order to improve the genetics of wheat that is able to adapt to tropical environments through hybridization, mutation induction, tissue culture, and genetic transformation. In vitro culture through somatic embryogenesis pathways plays an important role in genetic improvement and its integration with other breeding programs can positively affect the improvement of wheat quality, quantity, and development in Indonesia . The purpose of this study was to obtain an embryogenic callus induction method from the Dewata variety using five different types of explants, namely mature Seeds, immature embryos, immature seeds, leaf, stem, and to obtain combination of plant growth regulators and putrescine on somatic embryogenesis of wheat. The experimental design was prepared based on a complete randomized design with a combination treatment of embryogenic callus induction media consisting of 9 levels, namely : 1 ppm 2.4-D, 1 ppm 2.4-D + 1 ppm Picloram, ppm 2.4-D + 1 mg / L Picloram, 1 ppm 2.4-D + 10 -4 M Putrescine, 1 ppm 2.4-D + 1 ppm Picloram +10 -4 M Putrescine, 2 ppm 2.4-D + 1 ppm Picloram + 10 -4 M Putrescine, 1 ppm 2.4 D + 10 -3 M Putrescine, 1 ppm 2.4 D + 1 ppm Picloram + 10 -3 M Putrescine, 2 ppm 2.4 D + 1 ppm Picloram + 10 -3 M Putrescine. The results showed that the media used was able to induce embryogenic callus using mature seed and immature embryo, but immature seed and leaf were not able to produce embryogenic callus. The best media that produced the highest percentage of embryogenic callus was 2 ppm 2.4-D + 1 ppm Picloram + 10 -4 M Putrescine with as much as 85.9% in young embryo explants.


Introduction
Wheat (Triticum aestivum L.) is a cereal crop of a grain tribe native to the subtropics.
Wheat contains carbohydrates, a fairly high content of gluten and protein at 13.8 grams compared to 7.5 grams of rice and 9.2 grams of corn per 100 grams of ingredients.The consumption of foods derived from wheat and its derivatives is increasing beyond the consumption of tubers (Ariani, 2010).Indonesia's wheat imports from year to year are increasing, causing it to become the world's largest wheat importer country beating Egypt.
Wheat imports in 2020 reached 11.17 million tons (Central Statistics Agency, 2020).Food dependence from abroad is feared to be a threat to food security, especially wheat crops.
Wheat development is a challenge for Indonesia to produce wheat on a large scale in order to reduce wheat imports.Wheat cultivation in the highlands is limited, due to land competition with horticultural crops that have more selling value.Plant breeding programs need to be carried out in order to improve genetics and superior character (Lee & Huang, 2014;Hesami et al., 2018).Somatic embryogenesis if integrated with plant breeding programs such as mutation induction, somaclonal variation, protoplast fusion and genetic engineering can have a positive effect on improving the quality, quantity, and development of wheat in Indonesia (Komamine et al., 2005).
Many factors affect the success of somatic embryogenesis including growth regulators, types of explants, medium, and storage condition.Research on embryogenesis has been conducted by Setiawan (2015) which states that the use of more immature embryo (22.85%)produces embryogenic callus when compared to mature embryo (0.56%).Smit and Weijers (2015) states that auxin inducing promoters have reported that auxin spatial and temporal distribution mediate key steps in early embryo settings.Auxin is a regulator of the early add stages of somatic embryo establishment and postembryonic plant development, such as 2.4-D and picloram (Asghar et al., 2022).The use of polyamine compounds can increase the percentage of embryogenic callus.
Polyamine compounds commonly used are putrescine, spermine and spermidine (Aydin et al., 2016).The purpose of this study was to obtain an embryogenic callus induction method from the Dewata variety using five different types of explants, namely mature Seeds, immature embryos, immature seeds, leaf, stem, and to obtain combination of plant growth regulators and putrescine on somatic embryogenesis of wheat.

Methods
This research was conducted from June to October 2020 at the Network Culture Laboratory of the Faculty of Agriculture, Andalas University, Padang.The explants used were mature seeds, immature embryos, immature Seeds, leaves and stems.The explants were sterilized using fungicide solutions (mancozeb active ingredients) and bactericides (streptomycin sulfate active ingredients) with a concentration of 2 g/L each for 30 minutes, continued in laminar air flowcabinet (LAFC).Then, the explants were soaked in 1.25% of Na-hypochlorite solution for 30 minutes and rinsed with sterile aqueous.After that, the explants were soaked in 70% of alcohol for 3 minutes and rinsed with aqueous sterile 3 times while being stirred with a spatula and then transferred into a petri dish to proceed to the planting process.The medium used was MS (Murashige and Skoog) media, while other materials used were PGR 2,4-D, Picloram and Putrescine, sucrose 30 g/L, bacto agar 8 g/L and pH 5.8.
The design used was a Complete Randomized Design.The treatment used was a combination of plant growth regulator (PGR) and putrescine, which consists of 9 levels, namely: 2.4-D + 10 -4 M Putrescine, 1 mg / L 2.4-D + 1 mg / L Picloram +10 -4 M Putrescine, 2 ppm 2.4-D + 1 ppm Picloram + 10 -4 M Putrescine, 1 ppm 2.4 D + 10 -3 M Putrescine, 1 ppm 2.4 D + 1 ppm Picloram + 10 -3 M Putrescine, 2 ppm 2.4 D + 1 ppm Picloram + 10 -3 M Putrescine.The experiment used type f explants that were not on treatments.Each level of treatment was repeated 10 times, so that 90 experimental units obtained per explant were used.Observations include the percentage of callus formation, the time of callus induction, the percentage of embryonic callus formation, the fresh weight of the callus, the percentage of callus color, and the percentage of callus texture.
The data obtained were statistically analyzed using the STAR (Statistical Tool For Agriculture Research) program.Data were analyzed using Test F at a real level of 5%.Real different data was continued using the Duncan multiple hose test (DMRT) at a real level of 5%.

The percentage of forming callus
The combination of PGR and putrescine in Table 1 shows no influence on the percentage of explants forming the callus of wheat crops.The treatment given is able to induce 100% of callus in the explants of mature seed and immature embryos.This indicates a combination of PGR and putrescine used precisely to induce callus.The use of a combination of PGR and putrescine on immature seed and leaf explants has not been able to induce callus, while in stem explants, a combination of 2 ppm 2.4-D + 1 ppm Picloram + 10 -3 M Putrescine is able to induce the highest percentage of callus formation at about 20.00%.The success of explants in forming callus is influenced by several factors including the types of media and explant used.The use of explants and the right type will result in a high percentage of callus.Sharma et al. (2005) explained that different types of explants will give different responses.The immature embryo produced 94.00% of callus, the mature embryos produced 69% of callus and the scutellar did not induce callus.The use of putrescine was able to increase the percentage of callus formation.This is supported by Rakesh et al. (2021) that reported spermine and putrescine have effect on embryogenic calli development and meristemoid formation.

The timing of the callus induction
The results showed that the combination of PGR and putrescine had different influences.
Callus in mature seed appears in the range of 9.0-10.0day after culture (DAC), while in Immature Embryos, it ranges from 4.0 to5.3 DAC.On stem callus, it only appears with combination of 2 ppm 2.4 D+ 1 ppm Picloram + 10 -3 M Putrescine at about 16.6 DAC (Table 2)  The appearance of callus was characterized by the swelling of the explants, then a clear gellike layer appears on the surface of the explants.Young embryo explants give rise to callus faster when compared to other explants.This shows the use of young tissue is more effective because it has meristematic properties (still actively dividing).The PGR most commonly used for callus induction of wheat crops are 2,4-D, Picloram and NAA (Asghar et al., 2022).Plant growth regulators and type of explant play an important role in the initiation of callus because each explant requires a particular concentration of exogenous and endogenous hormones for callus production (Adhikari et al., 2013).

Percentage of embryogenic callus
The results showed that the influence of the combination of PGR and putrescine on the type of explants used was able to form embryogenic callus.Aged seed explants produced the highest embryogenic callus in a combination of 1 ppm 2.4-D +10 -4 M Putrescine by 30.0%, which was no different from a combination of 1 ppm 2.4-D + 10 -3 M Putrescine (20.0%).Meanwhile, in other combinations, embryogenic callus was not formed.In Immature Embryos, embryonic callus was formed in the range of 20.0-85.0%,while in young seed explants, the leaves and stems of embryogenic callus were not formed (Table 3).Embryogenic callus has a crumb texture, has a yellowish-white or yellow-white color with a green spot (Figure 1).The characteristics of embryogenic callus were also expressed by Tamimi and Othman (2021) report that a yellowish-yellow callus with a crumb texture.
The combination of PGR and putrescine given to young embryo explants was able to produce embryogenic callus in the entire combination.Meanwhile, the combination of 2 ppm 2.4-D + 1 ppm Picloram + 10 -4 M Putrescine produced the highest embryogenic callus with 85.0%.
The addition of putrescine can increase embryogenic callus and this is in line with what Aydin et al. (2016) report which stated that the addition of 10 -4 M putrescine results in 100% embryogenic callus in Kirik variety wheat.Several research reported that maximum callus induction for indica rice species was obtained on MS medium with 2-3 mg/L (Toppo et al., 2014;Abiri et al., 2017; followed by different lowercase letters in the column shows a noticeable effect based on the DMRT test level of α = 5 % followed by different lowercase letters in the column shows a noticeable effect based on the DMRT test level of α = 5 %

Table 1 .
Percentage of callus formation on some media and types of explant (%) Description: Data followed by different lowercase letters in the column showed a noticeable effect based on the DMRT test level of α = 5 %.

Table 2 .
Induction time of wheat callus on some media and explant type (DAC)

Table 3 .
Percentage of wheat embryogenic callus in some media and type of explans (%)