Evaluation of Hard Seed in Indonesia Local Mungbean (Vigna radiata L.)

The rate of hard seed in mungbean is a seed production and consumption and varies among varieties. The hard seed is characterized by the absence of pores in the epidermis and an impermeable seed coat created by the suberin layers, which is considered a significant issue for both to be correlated with the environment, seed size, and genetics. A germination test (ISTA rules) was used to determine the rate of the hard seed of 15 local mungbean genotypes collected by the Indonesian Agricultural Gene Bank. The seeds have been stored for three years at 18°C and 30-40% RH. The germination test using rolled paper consisted of 50 seeds with four replications. Hard seeds remained hard at the end of the germination test period because they had not absorbed water due to an impermeable seed coat. The hard seeds were then subjected to dormancy-breaking treatments, i.e., hot water rinsing, sandpaper scarification, soaking in H2SO4 98% and 50% for 30 minutes, and soaking in HCL 1N for 30 minutes. Local Sampang accession was found to have the highest number of hard seeds (15.5%). Lokal Sampang accession has a smaller seed, with an average seed length of 4.04±0.053mm and a seed width of 3.48±0,012 mm. Scarification with sandpaper was the most successful treatment to break the hard seeds’ dormancy, resulting in 100% germination.


Introduction
Mungbean is one of the essential food crops because mungbean has a high protein content [1]- [3].Mungbean is the most important legume group after soybean [4], [5].The origin of the mungbean is considered to be India.Domestication of mungbean is estimated in India about 3500 years ago, after that mungbeans spread to Asia and Africa [6].Indonesia is a country with a high level of mung bean diversity [4].There still needs to be a straightforward way to distinguish wild mungbean and landraces from domesticated ones, but cultivation has been selected for critical genetic changes [7].The diverse gene pool of local and wild mungbean comprises valuable genetic resources of beneficial genes that may help widen the genetic diversity of cultivated mungbean [6].Genetic distance analysis shows that landraces and cultivars will form subgroups according to their phenotypes [8].Exploration and conservation of mungbean from various areas in Indonesia is the first step of mungbean breeding.
Local accessions are a group that farmers rarely cultivate, so they are at risk of genetic erosion or extinction [9].In contrast, local accessions are a precious genetic resource in varietal assembly programs because they expand the genetic diversity of cultivated varieties [10].Local mungbean are often referred to as landraces.According to Villa 2005 [11], the definition of landraces is that it has a historical origin, has a distinct identity that has not undergone formal plant breeding, exhibits genetic diversity, is locally adapted, and is traditional.A dynamic crop population is associated with an agricultural system.The Indonesian Agricultural Gene Bank has explored and stored cultivated and local mungbean seeds in Indonesia.
Mungbeans are part of the Fabaceae family, which sometimes produce hard seeds.Hard seed do not germinate in ideal conditions for germination.Hard seed characterized by an impermeable seed coat, so seeds cannot perform imbibition and germination.There are differences in the structure of the seed coat by different varieties and species [6].The seed coat is the outermost part of the mature seed, sometimes also known as the testa.An illustration of the seed coat can be seen in the following picture [12].The role of the seed coat is to provide a protective layer for the developing zygote [13].The hard seed influenced by the environment, and it is is also an inheritable character [14].The hard seed advantage for seed plants is to keep persistent seed in the soil, spread germination over time, and the chance to establish the life cycle of plant species [15].The hard seed also maintains its existence and shelf life, and hard seeds resist disease and pests.On the other hand, hard seeds have a disadvantage in seed production because the hard seed will not germinate in the field.Hard seeds are also undesirable in mungbean's post-harvest and food processing.
This study aims to identify the presence of hard seeds in mung bean seeds collection that have been stored for more than three years in The Indonesian Agricultural Gene Bank.This study also aims to determine the method of seed scarification that can be used to treat hard seeds.

Materials
Seeds of 15 local mungbeans used in this study were provided by The Indonesian Agricultural Gene Bank.The seeds have been stored for three years at 18 o C and 30-40% RH.A germination test was carried out in the Seed quality test Laboratory of Indonesia Agricultural Gene Bank on August 2022.

Methods
Germination test (ISTA rules) [16] was used to determine the rate of the hard seed of 15 local mungbean genotypes collected by The Indonesian Agricultural Gene Bank.The seeds have been stored for over three years at 18 o C and 30-40% RH.The germination test using rolled paper consisted of 50 seeds with four replications.
Hard seeds remained hard at the end of the germination test period because they did not absorb water due to their impermeable seed coat.The hard seeds were then subjected to dormancy-breaking treatments, i.e., hot water rinsing, sandpaper scarification, soaking in H2SO4 98% and 50% for 30 minutes, and soaking in HCL 1N for 30 minutes.

Hard seed in Indonesia's local mungbean
This research shows that Local Sampang genotype of mungbean was found to have the highest number of hard seeds (15.5%).The following table (Table.1)shows the presence of hard seeds from 15 genotypes in the mungbeans collected from The Indonesian Agricultural Gene Bank.Hard seeds were found in Local Sampang and Local Pasar Kuningan, while no hard seeds were found in other genotypes.
The Local Sampang genotype has been collected from the Sampang region, district, and the regency seat of Sampang Regency in the East Java Province of Indonesia.The Regency is between 113°08' and 113°39' East Longitude and between 06°05' and 07°13' South Latitude.The Sampang area is situated around the equator.Regional boundaries bordering the Java Sea to the north.In the east, it is bordered by Pamekasan Regency.In the south, it is bordered by the Madura Strait.While in the west, it is bordered by Bangkalan Regency.
Recently, the local government of Sampang regency has recommended that farmers in Sampang cultivate mungbeans during the uncertain season.One of the high-yielding varieties suggested by the government is Vima.Local genotype also have the potential to be developed because they are more adaptive.However, this local accession has low productivity and longer life, and the harvest is not in unison.This study also shows that the local genotype of Sampang has a high percentage of hard seeds.
Seeds that do not germinate even though they are in a suitable environment are called hard seeds.Identification and assessment of the hard seed properties of mung bean plants are essential before breeding because this character varies greatly in different mungbean varieties.The presence of hard seeds in agricultural crop seed production is undesirable as they contribute to the emergence of heterogeneous seeds.In consumption seeds, these hard seeds are also unwanted because it has made it difficult in the cooking process.
The seed coat is one of the main determinants of seed germination, vigor, and longevity [17] because the seed coat is the primary modulator of interactions between the internal structures of the seed and the external environment [17].The seed coat and endosperm are the primary determinants of seed dormancy [13].Hard seed is a type of physical seed dormancy.Seed dormancy is essential to a plant's life history and strategy.Genetic and environmental influences and controlled hard seed [18].An elongated incision of a mungbean seed can be seen in Figure 2.  The seed coat is the primary determinant of seed germination.Based on studies conducted on legumes, seed size has a negative correlation with the presence of hard seeds in plants.The smaller green bean seeds have more hard seeds [18], [19].The length and width of the seeds have been measured with a caliper.Local Sampang genotype has smaller seeds, with an average seed length of 4.06mm and seed width of 3.48mm (Table 1).Particle volume and density are important in evaluating the quality of many technological processes and products.Seed volume was determined from the seed length dimension using a published equation.[19]: Volume = πLWT/6 (mm 3 ) Volume= (0.56822 VOLLW) + (0.05156 VOLLW 2 ) Volume= (0.46753 VOLLW) + (2.65493 × 10−5 VOLLW 2 ) with VOLLW = πLW 2 /6 Using the equation above, the volume of mungbean is obtained in Table 1.To validate the relationship between seed size and hard seeds, we estimated Pearson's correlation coefficient.According to the Pearson correlation in Table 2, a weak negative correlation exists between seed width (-0.27) and seed volume (-0.32) for hard seeds.A moderate negative correlation (-0,40) exists between seed length and hard seed.Seed size was negatively correlated with seed size components.Seed size is not a determining factor in the presence of hard seeds.However, from previous studies, hard seeds are found in small-sized seeds [20].The success of growing from large seeds that have been planted indicates a lower number of hard seeds [18].Hard seed -0,409 -0,271 -0,329 1

Scarification hard seed of Local Sampang mungbean
In this preliminary study, seed scarification has been carried out on the hard seeds of the remaining mungbeans from the germination test.We tried several scarification methods that are commonly used in seed technology to break dormancy.Due to the limitations of hard seeds each treatment only uses five hard seeds from the Local Sampang genotype of mungbean.a. Sandpaper scarification.Breaking dormancy by sandpaper scarification is mainly done on hard seeds.In physical dormancy, this method has a high chance of success because scratching the seeds with sandpaper can open the barrier on the seed coat so that imbibition can occur.Physical dormancy is broken when the seed coat forms small holes/water gaps through which water enters the seed [21].Mechanical scarification enhanced water absorption.All hard seeds from local Sampang can germinate into normal sprouts with this treatment (Figure 3).b.Soaking in H2SO4 50% Hard seeds were soaked in H2SO4 50% for 30 minutes, then washed to remove traces of sulfuric acid.All hard seeds of Local Sampang accession can normally germinate at this concentration.The duration of soaking and the concentration of sulfuric acid significantly affect the success of germination.c.Hot water rinsing Hot water rinsing also broke the dormancy of the hard seeds of mungbean.Based on previous research, soaking hot water and breaking dormancy also reduces pathogenic infections in mungbeans [22].d.Soaking in H2SO4 98% for 30 minutes, then washing to remove traces of sulfuric acid.
Soaking in a higher concentration of sulfuric acid can cause damage to the embryo [23].At high concentrations of sulfuric acid, abnormal sprouts were found, indicating that there had been damage to the embryonic level.Seed dormancy prevents the complete germination of intact and viable seeds, even under the right conditions ;water, temperature, light and dark and gaseous conditions [24].Physical dormancy caused by water impermeable seed coats and pericarp has been shown to occur in most angiosperms and is particularly common in legumes [25].Dormancy in mung beans is a physical dormancy that occurs only in the seed coat, not in the embryo Seeds with embryo dormancy (exogenous dormancy) require special environmental or chemical treatments [16].

Conclusion
The results of germination tests on the part of the mung bean collection owned by the Indonesia Agricultural Gene Bank showed that not all local genotypes had hard seeds.Of the 15 genotypes tested, only two genotypes had hard seeds: Local Sampang at 15.5% and Local Pasar Kuningan at 0.5%.Another local genotype has no hard seeds after storage for three years at 18 o C and 30-40% RH.Sandpaper scarification and soaking in H2SO4 50% were recommended treatments to break dormancy/hard seeds of mungbean.
e. Soaking in HCL 1N for 30 minutes Soaking Local Sampang seeds with 1N HCL failed in breaking the dormancy of the hard seeds of mung bean.Until the final count observations, all hard seeds have not germinated.It is estimated that the HCL concentration and duration of immersion are not appropriate.Further research is required to develop more effective dormancy-breaking treatments in mungbean.

Table 1 .
List of mungbean genotype and percentage of hard seeds

Table 2 .
Correlation coefficients between seed size and hard seeds