Selection of M3 mutant strains from 200 gy gamma irradiation on Cempo Ireng

. Climate change is a serious issue that can disrupt crop production and pest resistance, especially in rice crops. To tackle climate change, plant breeding can be employed to develop new characteristics that are resistant, using physical mutagenesis like gamma irradiation. Cempo Ireng is one type of rice in Indonesia with plant properties such as tall plant, long harvesting periods, and susceptibility to brown planthopper attacks. The research aims to obtain information and select M3 mutant strains with properties of shorter plants, early harvesting, and brown planthopper resistance. The study was conducted from February-July 2022 in Klaten Regency, Central Java, Indonesia. The research material consisted of 3 mutant strains of black rice M3(GH8'), M3(GH51'), and M3(GH52'), with the control variety of Cempo Ireng. The experimental design used was a Randomized Complete Block Design without replication. Data were analyzed descriptively and used t-test, at a significance level of 5%. The results showed that M3 mutant black rice had better growth and yield components than the control plant. Brown planthopper attacks of M3 mutant strains decreased to 24% compared to the control.


Introduction
Climate change is one of the challenges in agriculture that has a serious impact on food production.The threat of climate change is the increasing overall temperature which causes changes in the natural environment, affecting plants and animals natural patterns.The lack of adapting plants to climate change and changes in pest behavior is a catastrophic scenario for food production.The inability of plants to adapt to climate change leads to a decrease in yield due to a mismatch in plant requirements and conditions of plant growth.Also, changes in pest distribution can promote extensive pesticide application, threatening food safety [1][2][3][4].
Rice is the most widely consumed staple food commodity in Indonesia.There are several types of rice, including black rice.Black rice is usually chosen as an alternative to white rice due to superior nutritional content as anthocyanins [5].However, Cempo Ireng black rice has not been extensively cultivated because it has some drawbacks that are considered to result in low yield potentials such as long harvest period, tall plant height, relatively low yield potential, and low resistance to pest attacks [6].
Plant breeding help develop plant varieties for sustainable food production by generating new plant characteristics.One of the breeding techniques is gamma-ray irradiation.Gammaray induction is a physical mutagen that causes changes in DNA structure resulting in the emergence of improved characteristics of plants [7].Gamma-ray mutations change genotype structure, enhance species variation, and increase plant adaptability which is advantageous for plant breeding [8].The breeding program for Cempo Ireng black rice by gamma-ray irradiation helps emerges new traits in plants to cope with climate change, especially pest attacks.This study aims to obtain phenotype and pest resistance in shorter plant height and early flowering of M3 black rice for individual selection.

Phenotype of black rice
The study showed a difference in the flowering and harvesting period between M3 black rice in comparison to the control plants.Some of the individual plants of M3 have early flowering up to 7-9 days and harvesting up to 6-7 days (Table 1).The harvesting period of M3 rice showed that flowering characteristics of the black rice met the requirement of earlyharvesting rice.Early harvesting rice has a flowering period ranging from 105-124 days [9].Flowering is one of the characteristics that is affected by gamma-ray exposure.Gamma-ray exposure can cause changes in enzymes, proteins, and DNA related to flowering periods [10].
The results show that Gamma-ray irradiation causes different effects on the plant height.There is a difference in plant height in each of M3 as a result of gamma irradiation.M3(GH8') plant has the shortest plant with a minimum height of 100 cm with an average of 129,9 cm.Meanwhile, M3(GH51') plant has the lowest plant height of 117 cm with an average of 136,07 cm.Compared to the control plant with the lowest plant height of 115 cm with an average of 133 cm.The reduction in plant height M3(GH8') is more visible compared to M3(GH51') (Table 1).Gamma-ray irradiation can induce genetic mutation in plants, resulting in genetic variations.This genetic variation can be reflected in plants' physiology, one of which is plant height [11].The number of productive tillers of M3 black rice, namely M3(GH8') has the highest maximum value of productive tillers compared to the control plant which is 37 tillers.The average black rice productive tillers showed no significant difference.However, the highest average count of productive tillers is M3(GH8') with 17,5 tillers (Table 1).The results of productive tillers illustrate the number of rice tillers that produce panicles.The panicles that cannot form panicles during the vegetative stage are called unproductive tillers [12].The results on average panicle length showed a significant difference between M3 black rice compared to the control plant.Decreasing in panicle length of M3 black rice is due to the gamma-ray irradiation effect.The initial stages of gamma-ray irradiation affected more on quantitative plant characteristics, which lead to morphological abnormalities like plant height and panicle length [13].Gamma-ray irradiation causes plants to repair themselves due to chromosome damage leading to inhibition of plant growth [14].Decreasing of panicle length resulting in fewer grains formed on the plants.Irradiation causes a reduction in the number of filled grains due to alteration in cell structure related to carbohydrate translocation, indicating a decrease in carbohydrate translocation from roots to panicles [15].
The weight of 100 grains per panicle showed a significant difference between M3 black rice and the control plant.However, this result indicates a decrease in M3 grain shape compared to control plants.The weight of 100 grains represents grain shape, lower weight of 100 grains suggests a reduction in rice length.Slender grains have a heavier weight compared to round-shaped grains [16].The grain weight per plant depends on the number of rice grains formed on productive tiller, heavier grains means more rice grains formed.The average weight results show there is an increase and decrease in M3 black rice yield compared to control plants.The study showed that the percentage of plants infested by brown planthoppers increased each week.The final week of observation shows that the control plant has a 52% damage percentage, 39% on M3(GH8'), and 28% on M3(GH51') and M3(GH52').Lower in pest infestation percentage of M3 black due to genetic changes by gamma-ray irradiation.Gamma-ray irradiation can produce generations of plants more resistant to pest attacks compared to parental plants due to genetic improvement from gamma-ray irradiation treatment [17].Gamma-ray rice mutant improves pest resistance because gamma irradiation can help plants produce antimicrobial compounds, in rice plants by increase of phenolic acids [18].Brown planthoppers attack rice by piercing and sucking sap from the rice stem causing direct damage and facilitating rapid transmission of plant diseases [19].Prevention in brown planthopper feeding by increasing the hardness and thickness of the cell walls.Plant epidermis is considered to have an important role against insect attack, and determined brown planthopper feeding responses [20].Thick cell wall structure makes it more challenging for brown planthoppers to penetrate, effectively hindering their ability to feed from rice [21].Gamma-ray irradiation alters compounds in rice related to cell wall thickness, such as lignin [22].Lignin is a phenolic polymer found in the cell walls of vascular plants and mainly thickened secondary walls [23].High lignin content in rice plants enhances the thickness of the cell wall and increases the size of vascular tissue to make plant stems stronger and more resistant to stem-boring pests [24].

Individual selection
The results showed eight individual M3 black rice with shorter plant height and early flowering that were selected for M4 generation.Selected individuals of M3 black rice met the criteria of early harvest, short plant height, high productive tillers, and high weight per panicle (Table 4).

Table 1 .
Flowering time, harvesting time, plant height, and number of productive tillers of black rice.
Note : Numbers followed by a sign (*) are significantly different from the control as the results of Ttest (α=0.05).

Table 2 .
Panicle length and number of seeds per panicle.
Note : Numbers followed by a sign (*) are significantly different from the control as the results of Ttest (α=0.05).

Table 3 .
Weight of 100 seeds and weight seed per plant.