Seed vigour testing of Acacia mangium Willd. as a result of mutation

Plant breeding techniques are applied to increase the genetic variation of a plant. Induction of plant mutation using gamma irradiation is one of the methods that can be applied to improve the genetic diversity of plants. This study aims to analyze the vigour of mangium seeds treated by mutation. Mutation treatment was administered to M0 of mangium seed. This study used M2 mangium seeds and control mangium seeds (without mutation treatment). Several assays, such as growth, stress, and biochemical tests, were conducted to determine the mangium seed’s vigour. The growth test showed that M2 mangium seeds had higher germination values, germination height, dry weight, and vigour index than control mangium seeds. The stress test consisting of the Hiltner test, accelerated ageing test, and saturation test of the germination value of M2 mangium seeds wass higher than control mangium seeds. In the biochemical test, the electrical conductivity value of control mangium seeds was more significant than M2 mangium seeds. In general, M2 mangium seeds had higher vigour than control mangium seeds.


Introduction
The mangium plant (Acacia mangium Willd.) is a fast-growing plant most widely used in plantation development programs in the Asia and Pacific region [1].Mangium plants are easy to find in several regions in Indonesia because they do not have special conditions for growth.The use of mangium is very diverse and can be used for various industrial purposes.In general, mangium is used by Industrial Plantation Forests (HTI) to meet the demand for pulp and paper.Mangium is also used as a building material (beams, boards, rafters) and as raw material for the furniture industry to replace teak wood [2].The utilization of A. mangium plants is needed to fulfil several functions, such as production and protection, so the development of this A. mangium plant must be well-planned and can stabilize and improve the environment [3].
The remarkable growth of A. mangium plants must be considered since A. mangium is still in the seed state.Quality seeds can increase production regulated by the International Seed Testing Association (ISTA) based on testing standards for using seeds published nationally, regionally and internationally.Superior seeds can be obtained from guaranteed or glorified seed sources [4].
Plant breeding techniques have been widely applied to increase the genetic variation of a plant.One of the commonly applied breeding techniques is mutation.Mutation is a process in a gene that undergoes Changes in structure or genetic material that result in phenotypic changes are passed on to the next generation [5].Mutations in plant breeding aim to obtain new traits from an individual plant that can improve and increase genetic diversity.Induction of physical plant mutations by gamma irradiation is a method of plant breeding that can increase genetic diversity to support the selection of specific 1315 (2024) 012073 IOP Publishing doi:10.1088/1755-1315/1315/1/012073 2 characteristics in plant breeding [6].Applying gamma rays to seeds can penetrate up to several centimetres into the seed tissue and may damage the tissue through which it passes [7].The changes that occur can be utilized if energy such as gamma rays is given at the correct dose so that after the seeds germinate, the plants that grow will have the desired superior properties.One of the superior properties produced is the fast metabolism of seed cells to decompose germination-inhibitory chemicals [8].
Seed vigour can indicate a condition where the seed has good conditions so that the seed can grow if it is in the field and also shows how long the seed can be stored.Seed vigour tests can be carried out using several test methods, such as germination speed tests, accelerated ageing tests, electrical conductivity tests (Electrical Conductivity/EC), pressure tests, and biochemical tests.Seed vigour is described by two pieces of information about viability, namely the strength of growth and the storability of seeds, both of which can indicate the ability of seeds to grow into typical plants even though the field conditions are suboptimal or after the seeds reach a prolonged storage period [9].Therefore, seed vigour tests need to be carried out on seeds, especially those planted in the field, to minimize seed failure to survive and grow in the field.This study analysed the vigour of A. mangium seeds treated with mutations.

Time and Place
This research was conducted from January to March 2022.The research location was at the Silviculture and Greenhouse Laboratory of the Silviculture Division, Department of Silviculture, Faculty of Forestry and Environment, IPB.The accelerated ageing and electrical conductivity tests were carried out at the Seed Technology Laboratory, Department of Agronomy and Horticulture, IPB.

Tools and Materials
The tools used in this research were stationary, autoclave, 3000 mL plastic mica, solder, germinator, oven, 250 mL plastic mica, wire mesh, thermometer, analytical balance with an accuracy of 0.0001 g, sprayer, conductivity meter, jar bottle, ruler, camera, flashdisk, Microsoft Word 2019 software, Microsoft Excel 2019 software and SAS 9.0 software.
The materials used in this study were mangium seeds, zeolite media, aluminum foil, aquabides, distilled water, tissue, red bricks, 20 x 35 heat-resistant plastic, and rubber bands.

Seed Preparation
The seeds used in this study were mangium seeds, consisting of two types of seeds: mutated mangium seeds and non-mutated mangium seeds.The mutated mangium seeds were sourced from the Ciheuleut Seed Technology Research Institute (BPTP), Bogor.The mutation induction method was carried out on mangium seeds by gamma irradiation, namely by gamma-ray irradiation carried out at the National Nuclear Energy Agency (BATAN).The dose of gamma irradiation used is 150 grey.Mutational treatment was given to A. mangium M0 seeds.The M0 generation derivatives are referred to as M1 mangium seeds.Derivative M1 generation is referred to as M2 mangium seeds.The mutated mangium seeds used in this study were M2 mangium seeds.As a comparison, non-mutated mangium seeds (as control mangium seeds) were used from Jonggol (6°32' 41"S, 107°3'8"E), West Java.

Preparation of Germination Media
The germination medium used was zeolite sand.The size of the zeolite used is 1−2 mm.Before use, the zeolite was washed 6 times until the dirt and zeolite washing water were no longer cloudy.Next, the zeolite was dried on a plastic tarpaulin for 1 day.After drying, the zeolite was sterilized using an autoclave at 121°C for 15 minutes.The sterile zeolite was then put into a 3000 mL plastic mica container until the zeolite reached a height of 3 cm.
Brick fragments are used as additional germination media in the Hiltner test.The size of the brick fragments used is around 2-3 mm.Before use, the crushed bricks were sterilized by autoclaving at 121°C for 15 minutes.

Seed Preliminary Treatment
Before sowing, the seeds are given pre-treatment to break the dormancy of the seeds.Dormancy breaking was done by soaking in hot water (80 o C) for 1 minute, followed by plain water for 24 hours.After 24 hours, the next day, the seeds were sown into the sterilized zeolite medium.

Vigour Test
The vigour test treatments were (1) growth test: germination at optimum conditions (control), (2) stress test: saturation test, Hiltner test, rapid ageing test, and (3) biochemical test: electrical conductivity test.Each test consisted of 4 replicates, and each repetition consisted of 100 seeds planted.

Growth Test.
The growth test was carried out in the form of an optimum germination test which was used as a control.The zeolite media placed in the germination tub is doused with water until damp.Then the mangium seeds that have been broken by dormancy are planted by sowing them evenly on the surface of the media.The seeds were flattened over the entire media surface and then covered again with 0.5 cm of zeolite.The mangium seeds that have been sown are then placed in the greenhouse.

Stress
Test.Saturation Test.The mangium seeds were prepared in the same way as the optimum germination test (control).The seeds are then germinated in the germinator.The condition of the germinator is set without light, and the water reservoir inside the germinator is filled up to half of the volume of the water reservoir so that the humidity inside the germinator is maintained.Hiltner's test.The planting process in the Hiltner test was the same as in the optimum germination test (control).After that, the germination tub was again watered, and sterile brick fragments 3-4 cm high were added.After adding broken bricks, water the tub again to keep it moist.Accelerated ageing.Before being used up, the obsolescence box was filled with 100 mL of distilled water before being used up.Then the gauze is installed without touching the surface of the aquadest.Seeds were arranged on wire gauze, covered with tile cloth, placed in obsolescence boxes, and then tightly closed.Burning was done by incubating the seeds in the oven at 41 ± 0.3 °C and ± 100% RH.The seeds were incubated for 2 days without opening the oven.After 2 days, the seeds were removed and allowed to stand for 1 hour.Then the seeds were pre-treated and germinated on zeolite media.The germination method was carried out as in the optimum germination test (control), and the germination tanks were placed in the greenhouse.

Biochemical Test. Electrical Conductivity Test.
The seeds in the electrical conductivity test were not sown, and only the electrical conductivity of water was measured from the results of soaking the seeds.One hundred mangium seeds were weighed and then put into a glass jar. 100 mL of aquabidest was added to the jar containing the seeds, and then the glass jar was covered with aluminum foil.The glass jar containing the seeds was then stored in a room with a temperature of 20 o C−25 o C for 24 hours.After 24 hours, the electrical conductivity value was measured using a conductivity meter by dipping the conductivity rod on the conductivity meter into the glass jar for each repetition.Once this is done and the electrical conductivity value is known, the value is corrected for temperature and multiplied by the cell constant factor.The conductivity of water was measured with a conductivity meter and the results were expressed in µs cm g -1 [10].

Maintenance, Observation and Data Collection
Seed maintenance for optimum germination test (control), saturation test, Hiltner test and rapid obsolescence test was carried out for 1 month by watering the plants in the morning and evening and cleaning the weeds.Observations and data collection were carried out during the maintenance process for 1 month.The calculation of germinated seeds is carried out every day.The variables observed for the optimum germination test were germination power, germination height, dry weight and vigour index.
The variable observed for the saturation test, Hiltner test and rapid ageing test was germination power, while for the electrical conductivity test measurements were made on the immersion water of 4 several seed samples using a conductivity meter.The electrical conductivity was measured after measuring the conductivity value of the aquabidest immersion liquid containing the seeds, the pure aquabidest liquid (blank) and the weight of the seeds for each replicate.The formula measures the conductivity value.

EC=
Seed filled liquid conductivity value-seedless liquid conductivity value seed weight

Experimental Design and Data Analysis
The research design was carried out for each vigour test.The research design used a Completely Randomized Design (CRD) with one factor: the type of mangium seed.The mangium seed variety factor consisted of 2 levels: mangium seeds resulting from the M2 mutation and control mangium seeds (not the result of mutations).Each level consisted of 4 repetitions, and 1 repetition consisted of 100 seeds.The collected data were analyzed by ANOVA.The Least Significant Difference Test was carried out at a 95% confidence interval to determine the difference between treatments.

Result
The M2 mangium seeds had better vigour than the control mangium seeds, so the hypothesis was accepted.Seed vigour response is an interaction of the effects of mutations on seeds and the environment.Environmental factors in this study were considered uniform because the research was conducted in a greenhouse.
Table 1 shows that in the growth test, the type of A. mangium seed significantly affected germination power, germination height, dry weight and vigour index.Types of A. mangium seeds also significantly affect the results of stress tests (rapid ageing test and saturation test).In the biochemical test, the type of A. mangium seed significantly affected the conductivity value of the water soaked for A. mangium seed.Data on the Hiltner test were not analyzed statistics because there was only 1 replication that grew, making it impossible to do data processing.In the growth test, A. mangium M2 seeds had higher germination, germination height, dry weight and vigour index values compared to control A. mangium seeds.A. mangium M2 seeds had a germination rate 4 times higher than control A. mangium seeds (Figure 1A).The height value of M2 1315 (2024) 012073 IOP Publishing doi:10.1088/1755-1315/1315/1/0120735 mangium seed germination was 3 times greater than that of the control mangium seed (Figure 1B).On the dry weight value, M2 mangium seeds were 2 times higher than control mangium seeds (Figure 1C).The vigour index of M2 mangium seeds was 13 times greater than the control mangium seeds (Figure 1D).In the stress test, 3 types of vigour tests were performed on A. mangium seeds (Table 2).The results of the Hiltner test, accelerated ageing test, and saturation test showed that the germination rate of A. mangium M2 seeds had a higher value when compared to control A. mangium seeds.In Hiltner's test, A. mangium M2 seeds had a germination rate 60 times higher than control A. mangium seeds.In the obsolescence test, seeds mangium M2 had a germination rate 3 times higher than the control A. mangium seeds.In the saturation test, the germination value of A. mangium M2 seeds was 3 times higher than that of control A. mangium seeds.The dark conditions applied to the saturation test did not hinder the performance of the A. mangium seeds from germinating, only that the growing sprouts experienced etiolation.a Numbers followed by letters that are not the same indicate that there is a significant difference at the 95% confidence interval, x = the data is not statistically tested.
In the biochemical test, the mutation seeds had a lower electrical conductivity value when compared to the control seeds (Figure 2).This shows that the seeds resulting from the mutation have better seed quality compared to the control seeds

Discussion
Mutation treatment affected the vigour of A. mangium seeds so the hypothesis was accepted.Breeding mangium seeds had better vigour than seeds that had not been bred [4] or, in this study, were control seeds.
Morphologically, the M2 and control A. mangium seeds differed in size and weight.The M2 mangium seeds were more significant than the control mangium seeds (Figure 3).Large and heavy seeds contain more food reserves when compared to smaller seeds, and the embryo size is also larger [11].

B A
The results showed that the response of A. mangium M2 seeds to several variables such as germination rate, germination height and vigour index was higher than that of control A. mangium seeds, which means the M2 mangium seeds had better vigour than the control mangium seeds.One of the benchmarks for seeds having good vigour is having high germination [4].Genetic factors can also influence this significant difference between the two seeds.There was a significant difference in response to the storage period seeds in two different rubber plant clones due to the influence of different genetic factors [12].
The germination response in each vigour test, namely the stress test (Hiltner test, rapid aging test, saturation test) and biochemical tests showed that A. mangium M2 seeds had a higher germination rate than control A. mangium seeds.The vigour test for A. mangium seeds included vigour tests for vigour for growth and vigour tests for seed storage.The vigour test for growth which was carried out by applying the Hiltner test and saturation test, showed that the mutation given to A. mangium seeds increased the germination power even in environments with significant stress.Seeds germinating under a layer of shards or bricks in the Hiltner test will be able to describe the ability of seeds to withstand physical stress during germination.The vigour test of mangium seed storage was carried out by applying the rapid aging test [13].The high germination value in this test reflects that the seeds of A. mangium M2 can withstand high temperatures and humidity so that they can continue to grow well when germinated, which means that M2 mangium seeds had better seed vigour compared to control mangium seeds.Groups of seeds that have high vigour will still have high germination after going through the aging process, while groups of seeds that have low vigour will have reduced germination [14].Seed vigour can reflect the quality of a group of seeds used.Conducting an electrical conductivity test is one of the tests used to determine the physiological potential of mangium seeds related to seed vigour.The results showed that the conductivity value of the M2 mangium seeds was lower than that of the control mangium seeds.This shows that the seeds of A. mangium M2 have better seed quality and vigour compared to the control A. mangium seeds.The conductivity value of the seed describes the integrity of the seed cell membrane.Seed lots with the highest germination value have the lowest K ion leakage, so the higher the ion leakage level of a seed lot, the lower the vigour of the seed lot [15].Damaged seed cell membranes can result in leakage of sugar and electrolytes that affect the decrease in seed metabolism [16].
Mutations given to mangium seeds had better vigour compared to mangium seeds that were not given the mutation (control).It can be influenced by changes in genetic factors from seeds after being given a mutation.The mutation treatment was given to M0 mangium seeds, but the M2 generation used in this study still showed superior properties related to seed vigour.Soybean varieties of Wilis, Baluran, Burangrang, and Grobogan, which produced 1500 M2 lines from the results of embryogenic callus mutation induction of the four varieties.Through bulk and pedigree selection of the M2 soybean inbred lines tested in the field, 230 M4 inbred lines and 50 M5 inbred lines were obtained, which produced early maturing soybeans.Therefore, mutation techniques can be used in plant breeding to improve the desirable superior traits of A. mangium seeds.
The vigour test conducted in this study showed that the vigour of A. mangium M2 seeds was better than that of control A. mangium seeds.It was expressed in all vigour test results (growth test, stress test, and biochemical test) performed.The results of all tests were consistent, showing that the values of the variables observed in the M2 mangium seedling were higher than the control mangium seed.Based on these consistent results, all vigour tests conducted in this study can be carried out to determine a seed's vigour.The recommended vigour test to be carried out is a biochemical test, namely the electrical conductivity test.Environmental factors have a minimal effect when the electrical conductivity test is carried out.The conductivity test showed more accurate results for predicting the appearance of normal sprouts in greenhouses and nurseries [18].However, implementing the conductivity test requires a specific tool, namely a conductivity meter which can be an obstacle if the tool is unavailable.An alternative test that can be carried out to determine the vigour of A. mangium seeds is the growth test because it can be done without using special tools.

Conclusion
The M2 mangium seeds had a higher vigour than the control mangium seeds.In the growth test, the seeds of A. mangium M2 had a germination rate 4 times higher than that of the control A. mangium seeds.The height value of M2 mangium seed germination was 3 times greater than the control mangium seed.The M2 mangium seeds were 2 times higher than the control mangium seeds on the dry weight value.The vigour index of M2 mangium seeds was 13 times greater than the control mangium seeds.In the stress test, the Hiltner test results showed that the M2 seeds had a germination capacity of 60 times higher than the control A. mangium seeds.In the aging test, the germination rate of mangium M2 seeds was 3 times higher than that of the control mangium seeds.In the saturation test, the germination rate of mangium M2 seeds was 3 times higher than that of the control mangium seeds.For the biochemical test, the electrical conductivity value of the control A. mangium seeds was 4 times higher than that of M. mangium seeds.

Figure 2 .
Figure 2. Conductivity value of A. mangium seeds in biochemical tests.

Figure 3 .
Figure 3.Comparison of the size of the M2 mangium seeds (A) and the control mangium seeds (B).− = 0.5 cm.

Table 1 .
Recapitulation of the results of the analysis of the variety of A. mangium seeds which were treated with growth tests, stress tests, and biochemical tests.

Table 2 .
Germination of mangium seeds from the results of the Hiltner test, Accelerated ageing test, and saturation test.