Effect of Gamma Rays Irradiation to Cipedak Avocado Genetic Diversity

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INTRODUCTION

Avocado (Persea americana Mill.) is one of the world’s important fruit commodities. The fruit was included in family Lauraceae and the ordo Laurales. The plant is regarded as the “super food”

fruit because its nutritional contents and health benefits (Bhuyan et al., 2019; Kupnik et al., 2023).

World avocado production in 2019 exceeded from 7.18 million tons (Shahbandeh, 2021b). Indonesia is the 5^th largest avocado-producing country with a production of 461,610 tons (Shahbandeh, 2021a), of which however, only 0.05% that could be exported from its total production (BPS, 2019). Lack of superior variety, high heterogeneity of seedling, poor fruit quality, and discontinue fruits availability are the reasons for a very low overseas market access.

The Cipedak avocado is categorized as one of the superior avocados in Indonesia (Ditbenih, 2021) with preferred characters in variety reference for productivity, fruit size, peel color, flesh texture, fat and fiber contents (Kuswandi et al., 2017).

However, there are weaknesses character on this variety, like: thin skin, low flesh portion, and short fruit shelf life.

Plant breeding to improve the genetic characters in Cipedak avocados is needed to increase fruit production and quality to fulfil export market. Induction of mutation using gamma irradiation is one of the methods that could be implemented (Lamo et al., 2017mango, grapes etc. made the fruit breeders interested to breed through induced mutation. Since, mutations bring about variation, they provide the ultimate basis for evolution of new forms, varieties or species.

ARTICLE INFO Keywords:

Cipedak avocado DNA-RAPD

Gamma ray irradiation Leaf morphology Article History:

Received: January 28, 2023 Accepted: May 8, 2023

*⁾ Corresponding author:

E-mail: [email protected]

ABSTRACT

Avocado cv. Cipedak is a superior cultivar that match the preferences of Indonesian consumers. However, it has several weaknesses, such as low edible portion, thin skin, and short fruit shelf life. One effort to improve the character weakness is by mutation breeding using gamma ray irradiation. Induction of mutation by gamma ray irradiation at a dose of 10 Gy was conducted on Cipedak avocado shoots in October 2021 which produced 13 first generation avocado mutants (M1).

Observation on leaves morphological characters and DNA analysis were needed to find out the changes level of genetical diversity.

The research was conducted in Cukurgondang Research Station and Molecular Laboratory of Indonesian Tropical Fruits Research Institute from January to March 2022. The plant material used involved 13 M1 progenies and cv. Cipedak. The material used were 20 weeks old grafted plants. The observed variables were macro and micromorphological characters as well as DNA-RAPD analysis. The results showed that there were phenotypic and genotypic changes in 13 M1 avocados seedlings. The coefficients of differences on macro and micromorphological characters were up to 33% and 28%, respectively and the coefficient of difference for DNA-RAPD analysis was 74%.

ISSN: 0126-0537

Cite this as: Ihsan, F., Ashari, S., Soegianto, A., Sukartini, & Affandi. (2023). Effect of gamma rays irradiation to cipedak avocado genetic diversity. AGRIVITA Journal of Agricultural Science, 45(2), 231-249. http://doi.org/10.17503/agrivita.

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Effect of Gamma Rays Irradiation to Cipedak Avocado Genetic Diversity

Farihul Ihsan^1*), Sumeru Ashari²⁾, Andy Soegianto²⁾, Sukartini¹⁾, and Affandi¹⁾

1) Horticulture and Plantation Research Center, National Research and Innovation Agency, Cibinong, West Java, 16911, Indonesia

2) Department of Agronomy, Faculty of Agriculture, Universitas Brawijaya, Malang, East Java, 65145, Indonesia

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Mutations may result into deletion, inversion, translocation of chromosome and nucleotide base substitutions. Mutation can be induced artificially with the help of various physical and chemical agents which are called mutagens. Most commonly used Physical and chemical mutagens are gamma rays and EMS (Ethyl Methane Sulphonate;

Priyadarshan, 2019; Purwanto et al., 2019; IAEA, 2021). Mutation induction produces mutant plants rapidly, lead to change for better characters and retain the most of their original characters (Iwo et al., 2013; Harsanti & Yulidar, 2015; Gaswanto et al., 2016; Purba et al., 2021).

Induced mutation by Gamma ray irradiation to improve plant characters were reported in many plant species i.e. wheat (Di Pane et al., 2018;

Dwinanda et al., 2020), rice (Abdelnour-Esquivel et al., 2020), mango (Arthur, 2021) and avocado (El- mageid & Al-Kfrawey, 2018). Testing of plant genetic diversity could be done using morphological markers such as leaf macro-morphology, by observing its phenotypic appearance (Herison et al., 2018;

Tahir et al., 2019; Ihsan & Santoso, 2020). Micro- morphological observations are needed if macro- morphological observations cannot distinguish the diversity. Leaf micro-morphological characteristics are the structure of microscopic parts of the leaf, such as the shape, type, and arrangement of cells on the leaf surface (Hanum et al., 2013;

Husnudin et al., 2019; Samiyarsih et al., 2020).

However, this marker is strongly influenced by the environment, hence, other markers that are free from environmental influences are needed, namely DNA molecular markers. DNA molecular diversity testing could be done by using DNA-RAPD analysis (Zulfahmi et al., 2015; Prihartini et al., 2016; Avivi et al., 2019; Poornima et al., 2020).

Induced mutation by several doses of Gamma irradiation had been reported on avocado cv. Hass and Fuerte. The results revealed that vegetative growth parameters were significantly reduced by the increasing of Gamma-ray doses.

Unlike the vegetative growth parameters, the oil contents increased by the increasing of Gamma- ray doses (El-mageid & Al-Kfrawey, 2018). Mutation induced by gamma ray irradiation with dose of 10 Gy to Cipedak avocado scion had been carried out in October 2021 and generated 13 mutant seedlings for first generation (M1) (Ihsan et al., 2022). To determine the effect of gamma irradiation on plant genetic changes, especially on fruit characters, considerable effort is required. This is because it is

necessary to have the results of propagation and maintenance of all M1 plants, so that the plants can be observed. Based on this, it is necessary to do early selection in the seed phase to reduce costs. Early selection can be done by observing the morphological and molecular characters such as DNA-RAPD analysis. In molecular testing, it is necessary to know the profile of the electrophoretic results which are expected to represent the quality character of the intended fruit. Therefore, it is necessary to recognize the differences in these characters from the comparison varieties. The SLT and SLP comparison varieties had medium skin thickness and small seeds, while the Raja Giri and Tongar varieties had medium skin thickness and seed sizes. The Siginjai variety is classified as a type of avocado that has thick skin and large seed sizes, while Cipedak has the character of a fruit with a thin skin, but with large seeds. This study aims to determine the effect of gamma irradiation on genetic changes in Cipedak avocados. In addition, this research can be used as a basis for early selection of M1 avocados in the seed phase.

MATERIALS AND METHODS

This study was conducted from January to March 2022. Study of leaf morphological characterization was conducted in Cukurgondang Research Station, Pasuruan Regency, meanwhile DNA-RAPD analysis was conducted in Molecular Laboratory of Indonesian Tropical Fruit Research Institute (ITFRI), located in Sumani, Solok regency, West Sumatra.

Thirteen first-generation mutants (M1) of avocado seedlings previously irradiated by gamma rays and one Cipedak avocado seedling were used as research materials. Those plants were 20 weeks old after grafting. M1 avocado scions were irradiated by gamma rays at dose of 10 Gy and then were grafted onto rootstocks. M1 avocado seedlings were coded as: (1). E10-I-201, (2). E10-I-204, (3).

E10-I-206, (4). E10-I-209, (5). E10-II-211, (6). E10- II-213, (7). E10-II-215, (8). E10-II-218, (9). E10- II-219, (10). E10-III-260, (11). E10-IV-268, (12).

E10-IV-279, and (3). E10-V-294. The plant material for examination and RAPD primer selection were leaves of 6 avocado varieties like SLT, SLP, Raja Giri, Tongar, ST5RRRiginjai and Cipedak.

Research Activities

The observations were made on 14 treated avocado seedlings consisting of 13 gamma-

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irradiated M1 and one cv. Cipedak. The M1 avocado scions were irradiated by gamma rays at dose of 10 Gy and then were grafted onto the rootstocks. The avocado scions of 10-15 cm in length were taken from a branch of the Cipedak parent tree that was healthy and not in the flush period. The leaves on the shoots are discarded, and the shoots are packed in black plastic bags. Furthermore, the shoots in black bags were irradiated with gamma rays at a dose of 10 Gy. After irradiation, the grafts were grafted onto the rootstock.

The rootstocks are local avocado seeds originated from the collectors. Selection of rootstock candidates is carried out in 2 stages. The first selection is made on the seeds before sowing. The seeds to be sown are those selected the weight more than 65 grams, healthy and not attacked by pests and diseases. Selected avocado seeds were sown in 20 x 30 polybags with 1:1 soil and manure media. The second selection was made when the seeds reached 4 months old. The criteria for the selected rootstock candidate were vigor, not attacked by pests and diseases, a minimum height of 50 cm, and a stem diameter near the soil surface of at least 8 mm.

The grafting method used to make grafted seeds is a wedge or cleft graft. After 9 weeks, the grafted seeds were transferred into a 40 x 50 polybag containing soil and manure 1:1. Furthermore, grafted avocado seeds were maintained under UV plastic shade and 45% paranet for 11 weeks. Plant maintenance were carried out through watering once in 2 days and spraying insecticides and fungicides to prevent pest and disease attacks.

Leaf Macro-morphology Observation

Macro-morphological observations of leaves used avocado descriptors from the International Institute of Plant Genetic Resources (IPGRI, 1995) and the Directorate of Horticultural Nurseries (Ditbenih, 2013). Observations were made on three optimal leaf samples (4^th to 6^th from the apical).

The variables observed were leaf shape, leaf base shape, leaf tip shape, leaf blade twist, leaf tip twist, maternal leaf bone arch, leaf shape in cross section, leaf shape in longitudinal slice, petiole length, leaf length, leaf width, fine hairs on the lower leaf surface, fine hairs on the upper leaf surface, color of young leaves/ shoots, color of old leaves, cross angle of petioles, and wave of leaf edges. These parameters were listed on IPGRI

and Ditbenih descriptors which are usually used to differentiate the diversity of avocado accessions.

Leaf Micro-morphology Observation

Leaf micro-morphological characters were observed from 3 optimal leaves that were located at the 4^th to 6^th leaves from the shoot tip. The observations were made under a microscope with magnification up to 250 times. The preparations were made using the printing technique (Ihsan

& Marta, 2016). Each observation spot was photographed using a digital camera. The observed variables were density, length and width of stomata as well as density and length of trichome.

DNA-RAPD Analysis

The CTAB method was used to prepare DNA templates for PCR reactions. The primers used were the result of a selection of 50 primers from the ITFRI collection (Table 1). These primers were used to amplify six avocado accessions with very conspicuous character of skin quality and seed size, namely SLT, SLP, Raja Giri, Tongar, Siginjai and Cipedak. The primers that successfully amplified the DNA of the six avocado accessions will be used for the PCR reaction of M1 mutants and their comparison (Cipedak).

Data Analysis

Principal Component (PC) analysis with SPSS 17 software was used to determine the contribution of qualitative and quantitative characters to the diversity of avocado accessions. The data were presented descriptively and translated into binary data, a value of 1 for the expressed phenotype and a value of 0 for the unexpressed phenotype. Similarly, in the DNA-RAPD analysis, the analytical data were transformed into binary data which determined based on the appearance of the bands, the value of 1 for the presence of the band and the value of 0 for the absence of the band. Morphological binary data and DNA-RAPD data were further analysed using NTSYS 2.02 computer software to produce a dendogram that was formed using the Dice-Sorensen similarity coefficient along with genetic distance between groups and Unweighted Pair Group Method with Arithmetic (UPGMA). The dendogram would show the genetic similarity or difference relationship that is displayed in the form of a genetic similarity dendogram. The similarity coefficient uses a value scale of 0 to 100%. A value of 100% indicates a 100% similar genotype.

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Table 1. Amplification of 50 DNA-RAPD primers on 6 avocado varieties (SLT, SLP, Raja Giri, Tongar, Siginjai and Cipedak)

No. Primer Basa arrangement 5’-3’ Polymorphic (%)

1 OPA-01 CAG GCC CTT C 50.00

2 OPA-02 AGT CAG CCA C 33.33

3 OPA-03* AGT CAG CCA C 66.66

4 OPA-04 AAT CGG GCT G 16.66

5 OPA-07 GAA ACG GGT G 0.00

6 OPA-08 GTA ACT TAG G 0.00

7 OPA-10 GTC ATC GCA G 50.00

8 OPA-11 CAA TCG CCG T 0.00

9 OPA-13* CAG CAC CCA C 100.00

10 OPA-14 TCT GTG CTG G 50.00

11 OPA-17 GAC CGC TTG T 0.00

12 OPA-18 AGG TGA CCG T 16.66

13 OPA-19 CAA ACG TCG G 33.33

14 OPAV-6 CCC GAG ATC C 50.00

15 OPB-01 GTT TCG CTC C 50.00

16 OPB-18 CCA CAG CAG T 0.00

17 OPC-01 TTC GAG CCA G 0.00

18 OPC-04 CCG CAT CTA C 33.33

19 OPC-08 TGG ACC GGT G 16.66

20 OPC-09 CTC ACC GTC C 16.66

21 OPC-12 TGT CAT CCC C 50.00

22 OPC-14* TGT CAT CCC C 66.66

23 OPC-16 CAC ACT CCA G 0.00

24 OPC-19 TTC CCC CCA G 0.00

25 OPC-20 ACT TCG CCA C 50.00

26 OPF-13 GGC TGC AGA A 33.33

27 OPG-01* CTA CGG AGG A 66.66

28 OPJ-01 CCC GGC ATA A 33.33

29 OPK-03 CCC TAC CGA C 16.66

30 OPM-16 GTA ACC AGC C 16.66

31 OPM-20 AGG TCT TGG G 16.66

32 OPN-03 GGT ACT CCC C 50.00

33 OPN-09 TGC CGG CTT G 33.33

34 OPN-12 CAC AGA CAC C 0.00

35 OPP-08 ACA TCG CCC A 0.00

36 OPS-12 CTG GGT GAG T 0.00

37 OPX-15* CAG ACA AGC 66.66

38 OPX-17 GAC ACG GAC C 16.66

39 OPY-15* AGT CG CCC TT 83.33

40 RAPD-1 GGT GCG GCG GAA 16.66

41 RAPD-2 GTT TCG CTC C 50.00

42 RAPD-3 GTA GAC CCG T 16.66

43 RAPD-4 AAG AGC CCG T 33.33

44 RAPD-5* AAC GCG CAA C 83.33

45 RAPD-6 CCC GTC AGC A 50.00

46 UBC-811* GAG AGA GAG AGA GAG AC 83.33

47 UBC-817* CAC ACA CAC ACA CAC AA 100.00

48 UBC-820* GTG TGT GTG TGT GTG TT 83.33

49 UBC-826* ACACACACACACACACC 100.00

50 UBC-834* AGA GAG AGA GAG AGA GYT 83.33

Remarks: * = chosen DNA-RAPD primer

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RESULTS AND DISCUSSION Leaf Macro-morphological Character

A varied data was reached among 13 M1 avocado gamma ray irradiation seedlings and Cipedak avocado seedling on qualitative and quantitative parameters of leaf macro-morphological character. Macro-morphological qualitative characters of leaves with the same and various traits from all accessions were presented in Table 2 and Table 3, respectively.

The result of quantitative observations for the variables of petiole length, leaf blade length and leaf blade width in the tested accessions were displayed in Table 4. The longest and the shortest petiole length were given by Cipedak (3.00 cm) and E10- II-219, respectively, while the longest and shortest leaf blade lengths were presented by Cipedak (16.78 cm) and E10-IV-279 (11.15 cm), respectively. The widest and narrowest leaf blade width was exhibited by Cipedak (6.60 cm) and E10-II-219 (4.35 cm), respectively. PC analysis results show that the variables of petiole length, leaf blade length and leaf blade width contribute to the diversity of avocados (Table 5). The macro-morphological performance of leaves was shown in the Fig. 1 and the dendogram of similarity grouping based on macro- morphological characters was presented in Fig. 2.

The dendogram of 14 tested avocado accessions based on macro-morphological characters showed genetic similarity with a coefficient of 67% to 89%.

The similarity grouping characters was divided into 4 groups. Group I consisted of eight accessions, i.e:

E10-I-201, E10-III-260, E10-I-204, E10-I-209, E10- IV-268, E10-II-213, Cipedak, E10-II-211. Group II of three accessions, i.e: E10-I-206, E10-II-218, and E10-II-219. Group III of two 2 accessions, i.e:

E10-II-215 and E10-IV-279, and Group IV of one accession, i.e: E10-V-294.

In quantitative characters, the average leaf petiole length was longer in group I (2.31 cm)

then group III (2.07 cm), group II (1.78 cm) and the shortest in group IV (1.75 cm). The average leaf blade length was longer in group I (15.51 cm), then group III (12.67 cm), group IV (12.65 cm) and the shortest in group II (11.67 cm). The widest leaf blade width was in group I (5.97 cm), then group III (5.57 cm), group IV (5.2 cm) and narrowest in group II (4.54 cm). The qualitative distinguishing characters of the 4 groups are: leaf blade shape, leaf tip shape, and maternal leaf bone arch. Leaf blade shape characters are oval in group I, II and IV, and narrowly obovate in group III. The leaf tip shape was acute to intermediate in groups I and II, intermediate in group III and acute in group IV. The characteristics of the maternal leaf bone arch are flat, arch to leaf base arch in group I, flat in group II and III, and leaf bases arch in group IV.

Gamma ray irradiation on avocado seedlings with a dose of 10 Gy was able to induce mutations and change the phenotypic properties of the macro-morphological characters of the leaves. The results proved that 6 out of 13 qualitative character variables were changes. The observations of quantitative character variables were also exhibited a change in size. The comparation of seedling between M1 mutans and Cipedak as a control was indicated that treated seedlings were decrease in the variable of petiole length, leaf blade length, and leaf blade width. The changes in the size of plant organ to become shorter due to mutations caused by gamma ray irradiation also occurred in the study of mutations in winged-bean (Saragih et al., 2020), sunflower (Monikasari et al., 2018), leunca (Saragih & Aisyah, 2019), avocado (El-mageid & Al- Kfrawey, 2018), and mangosteen (Widiastuti et al., 2010). The dendogram of 14 avocado accessions that were tested based on macro-morphological characters showed phenotypic differences up to a coefficient of 33%.

Table 2. Leaf macro-morpological caracters with the same traits of 13 M1 accessions and 1 Cipedak avocado accession

Leaf character variable Character

Leaf base shape Acute

Leaf blade twist Untwist

Longitudinal slice shape Flat

Fluffy hairs on the lower surface of leaf Sparse

Fluffy hairs on the upper surface of leaf Sparse

Colour of shoots Greenish red

Petiole crotch angle Acute

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Table 3. Leaf qualitative macro-morphological characters of 13 M1 accessions and 1 Cipedak avocado accession Leaf charactersCipedakE10-I-201E10-I-204E10-I-206E10-I-209E10-II-211E10-II-213 Leaf blade shapeOvalOvalOvalOvalOvalOvalOval Leaf tip shapeAcute; intermediateAcute; intermediateAcuteAcute; intermediateAcute; intermediateAcuteAcute; intermediate Maternal leaf bone archFlat; leaf base archFlat; archFlatFlatFlat; archFlatFlat; leaf base arch Leaf shape in cross section

Flat to slightly concave

Intermediate concave; very concave

Flat to slightly concave; Intermediate concave

very concaveFlat to slightly concave Color of old leavesGreen; dark greenDark greenGreenDark greenGreenGreenGreen; dark green Wave of leaf edgesFlat; one side wavyOne side wavyFlatWavyFlat; wavy

One side wavy; wavy Flat; one side wavy; wavy

Leaf charactersE10-II-215E10-II-218E10-II-219E10-III-260E10-IV-268E10-IV-279E10-V-294 Leaf blade shapeNarrowly obo- vateOvalOvalOvalOvalNarrowly obovateOval Leaf tip shapeIntermediateAcute; intermediateAcuteAcuteAcuteintermediateAcute Maternal leaf bone archFlatFlatFlatFlat; leaf bases archyFlat; leaf bases archyFlat Leaf bases archy

Leaf shape in cross section

Intermediate concave; very concave

Flat to slightly concave; Intermediate concave

Flat to slightly concaveFlat to slightly concaveFlat to slightly concave Color of old leavesGreenGreenGreenDark greenGreenGreenDark green Wave of leaf edgesOne side wavyWavy

Flat; one side wavy; wavy

Flat; one side wavy

One side wavy;wavy

One side wavyFlat

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Fig. 1. Macro-morphological performance of leaves as a result of gamma irradiation; (A) Cipedak, (B) E10-I-201, (C) E10-I-204, (D) E10-I-206, (E) E10-I-209, (F) E10-II-211, (G) E10-II-213, (H) E10-II-215, (I) E10-II-218, (J) E10-II-219, (K) E10-III-260, (L) E10- IV-268, (M) E10-IV-279, (N) E10-V-294

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Fig. 2. Dendogram based on macro-morphology of avocado leaves

Table 4. Macro-morphological characters of petiole length, blade length and leaf blade width of 13 accessions M1 and 1 Cipedak avocado accession

Accessions Leaf petiole length (cm) Leaf blade length (cm) Leaf blade width (cm)

Cipedak 3.00 ±0.21 16.78 ±0.48 6.60 ±0.87

E10-I-201 2.35 ±0.39 14.81 ±0.88 6.01 ±0.53

E10-I-204 2.25 ±0.15 15.40 ±0.40 5.75 ±0.15

E10-I-206 1.95 ±0.08 11.60 ±0.52 4.58 ±0.20

E10-I-209 2.20 ±0.52 14.97 ±0.64 5.55 ±0.47

E10-II-211 2.20 ±0.30 16.70 ±0.20 6.50 ±0.50

E10-II-213 2.54 ±0.43 16.16 ±0.72 6.38 ±0.48

E10-II-215 2.35 ±0.05 14.20 ±0.40 5.75 ±0.05

E10-II-218 1.81 ±0.17 11.45 ±0.86 4.70 ±0.55

E10-II-219 1.60 ±0.10 11.98 ±0.48 4.35 ±0.09

E10-III-260 2.10 ±0.36 14.93 ±1.69 5.53 ±1.11

E10-IV-268 1.90 ±0.10 14.40 ±0.40 5.45 ±0.35

E10-IV-279 1.80 ±0.00 11.15 ±0.35 5.40 ±0.10

E10-V-294 1.75 ±0.05 12.65 ±0.15 5.20 ±0.20

Table 5. Vector values of 3 quantitative macro-morphological characters on the two main components of 13 M1 accessions and 1 Cipedak avocado

Accessions Main Component

PC1 PC2

Leaf petiole length 0.929 0.365

Leaf blade length 0.949 -0.241

Leaf blade width 0.960 -0.115

Remarks: Component values written in bold are character values that have an effect (value ≥ 0.75)

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Leaf Micro-morphological Characters

Diverse micro-morphological characters of the leaf variable such as stomatal density, length, and width, as well as trichome density and length were observed among the tested accessions. A complete data of leaf variable was presented in Table 6. The dense and sparse stomatal densities were exhibited by E10-IV-268 (544.00 unit/ mm²) and E10-I-204 (388.00 unit/ mm²), respectively. The longest and shortest stomata lengths were denoted by 1.78 µm at E10-II-213, E10-III-260 and 1.72 m at E10-I-204, E10-I-206, respectively. The widest (1.81 µm) and narrowest (1.73 µm) stomata width were pointed by E10-I-201, E10-I-206 and E10- II-218, respectively. The dense trichome density was displayed by Cipedak (45.08 strands/ mm²) and the sparse was shown by E10-III-260 (12.47 strands/mm²). The longest trichome length was presented by Cipedak (12.83 µm) and the shortest trichome was shown by E10-II-215 (8.51 µm).

Among 14 avocado accessions were tested based on micro-morphological characters indicated

that they shared genetic similarity with coefficient of 72% to 100% and grouped into 4 cluster. A complete genetic similarity dendogram and micro- morphological performance of the leaves were presented in Fig. 3 and Fig. 4. The grouping of similarity characters was clustered into 4 groups with the following details. Group I has 5 accession members, i.e: Cipedak, E10-I-209, E10-II-211, E10- II-213 and E10-I-206. Group II has 6 accessions, i.e: E10-I-204, E10-II-215, E10-IV-279, E10- II-219, E10-V-294, and E10-IV-268. Group III has 2 accessions, i.e: E10-I-201 and E10-III-260 and group IV has 1 accession, E10-II-218. The results of the PC micro-morphological characters analysis are shown in Table 7. Variables of trichome density and length contributed to avocado diversity with an eigenvector value > 0.75. This is in accordance with the opinion (Hadiati et al., 2021), which states that if the PC value is more than 0.75 then the observed character components are the difference between groups.

Table 6. Leaf surface micro-morphological characters Accessions Stomata density (pieces/ mm²) Stomata length

(µm) Stomata width

(µm) Trichome density

(strands/ mm) Trichome length (µm) Cipedak 414.00 ±41.61 1.77 ±0.01 1.80 ±0.00 45.08 ±8.21 12.83 ±6.58 E10-I-201 430.67 ±61.23 1.75 ±0.00 1.81 ±0.01 20.50 ±3.65 8.67 ±0.51 E10-I-204 388.00 ±60.00 1.72 ±0.02 1.77 ±0.02 28.38 ±5.18 8.95 ±0.05 E10-I-206 497.67 ±36.61 1.72 ±0.02 1.81 ±0.01 34.38 ±8.68 9.94 ±0.65 E10-I-209 479.33 ±94.70 1.77 ±0.01 1.80 ±0.00 35.15 ±7.47 8.56 ±0.19 E10-II-211 438.67 ±40.26 1.76 ±0.01 1.80 ±0.00 28.75 ±4.21 9.55 ±1.35 E10-II-213 403.11 ±12.09 1.78 ±0.03 1.79 ±0.01 26.88 ±5.16 9.48 ±0.28 E10-II-215 440.00 ±28.00 1.77 ±0.02 1.76 ±0.03 26.49 ±2.85 8.51 ±1.71 E10-II-218 533.33 ±22.03 1.75 ±0.00 1.73 ±0.01 44.61 ±3.91 10.15 ±1.46 E10-II-219 456.00 ±28.00 1.75 ±0.00 1.77 ±0.02 37.95 ±10.09 9.36 ±0.87 E10-III-260 468.00 ±76.21 1.78 ±0.01 1.80 ±0.02 12.47 ±6.36 10.09 ±2.07 E10-IV-268 544.00 ±8.00 1.75 ±0.02 1.77 ±0.02 37.19 ±10.03 9.61 ±1.30 E10-IV-279 446.67 ±33.54 1.76 ±0.03 1.75 ±0.00 29.75 ±7.14 8.52 ±0.68 E10-V-294 494.93 ±57.82 1.76 ±0.01 1.76 ±0.01 38.46 ±11.32 9.48 ±0.42 Table 7. Vector values of 5 quantitative macro-morphological characters on the two main components of 13 M1 accessions and 1 Cipedak avocado

Accessions Main Component

PC1 PC2

Stomata density 0.700 -0.174

Stomata length -0.338 0.328

Stomata width -0.641 0.452

Trichome density 0.857 0.329

Trichome leghth 0.260 0.923

Remarks: Component values written in bold are character values that have an effect (value ≥ 0.75)

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Quantitative character average trichome density was highest in group II (38.98 strands/mm²), followed by group IV (38.46 strands/mm²), group I (29.3 strands/mm²) and the lowest was group

III (28.12 strands/mm²). The longest quantitative average trichome length character was in group II (9.81 µm), followed by group I (9.71 µm), group IV (9.48 µm) and the shortest was group III (8.51 µm).

Fig. 3. Dendogram based on micro-morphology of avocado leaves

Remarks: → = Stomata. → = Trichome

Fig. 4. Micro-morphological performance of Cipedak leaves (left) and E10-II-218 (right)

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Gamma ray irradiation in avocado with a dose of 10 Gy was also able to induce mutations and change the phenotypic properties of the micro- morphological characters of the leaves. The results showed a change in the character of 5 observed variables. Changes in stomata density in 13 M1 seedlings compared to Cipedak seedling occurred randomly indicated by denser and sparser stomata.

The denser stomata density of M1 seedlings were occurred at: E10-I-201, E10-I-206, E10-I-209, E10- II-211, E10-II-215, E10-II-218, E10-II -219, E10- III-260, E10-IV-268, E10-IV-279, and E10-V-294.

While, the sparser stomatal density were occured at E10-I-204 and E10-II- 213. The dendogram of 14 avocado accessions were tested based on micro-morphological characters showed genetic differences coefficient up to 28%.

DNA-RAPD Analysis

The amplification of 50 DNA-RAPD primers were tested on 6 avocado varieties, namely: SLT, SLP, Raja Giri, Tongar, Siginjai and Cipedak.

Selection of primer was based on polymorphic amplification of more than 50% of the total varieties tested, so that 12 selected primers were obtained, i.e: OPA-01, OPA-13, OPC-14, OPG-01, OPX-15, OPY-15, RAPD- 5, UBC-811, UBC-817, UBC-820, UBC-826, and UBC-834.

Table 8 showed the electrophoretic amplification and fragment size of 13 avocado accessions of M1 and cv. Cipedak using 12 selected DNA-RAPD primers. Accessions that resulted in polymorphic amplification of all primers were:

Cipedak, E10-I-209, E10-II-211, and E10-IV-268.

Accession E10-I-201 resulted in polymorphic amplification on 11 primers, E10-I-204 polymorphic on 6 primers, E10-I-206 polymorphic on 9 primers, E10-II-213 polymorphic on 2 primers, E10-II-215 polymorphic on 2 primers. 10 primers, E10-II-218 polymorphic on 6 primers, E10-II-219 polymorphic on 3 primers, E10-III-260 polymorphic on 9 primers, E10-IV-279 polymorphic on 8 primers, E10-V-294 polymorphic on 9 primers.

Table 8. Electrophoretic amplification and fragment size of 13 M1 accession and 1 Cipedak avocado accession to 12 DNA-RAPD primers

Accessions Primer Amplification Fragment size (bp)

Lower Upper

Cipedak

OPA-03 Polymorphic 750 1250

OPC-14 Polymorphic 625 1250

OPG-01 Polymorphic 300 1750

OPX-15 Polymorphic 500 1500

OPY-15 Polymorphic 300 875

RAPD-5 Polymorphic 500 1000

UBC-811 Polymorphic 500 1250

E10-I-201

UBC-817 Monomorphic 1000 1000

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Table 8. (continued)

Lower Upper

E10-I-204

OPA-13 No amplification - -

OPC-14 No amplification - -

OPG-01 No amplification - -

OPX-15 No amplification - -

RAPD-5 No amplification - -

E10-I-206

OPY-15 Monomorphic 875 875

E10-I-209

E10-II-211

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Lower Upper

E10-II-213

OPX-15 No amplification - -

OPY-15 No amplification - -

UBC-817 No amplification - -

E10-II-215

OPX-15 Monomorphic 1500 1500

E10-II-218

E10-II-219

OPA-13 Monomorphic 625 625

OPX-15 Monomorphic 1000 1000

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Lower Upper

E10-III-260

E10-IV-268

E10-IV-279

OPC-14 Monomorphic 1250 1250

E10-V-294

OPC-14 Monomorphic 1250 1250

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Table 9 showed the percentage and recapitulation of the fragment size of 12 DNA-RAPD primers were tested on 13 M1 avocado and cv.

Cipedak. The amplification resulted in DNA bands that were polymorphic ranging from 50–92.85 %, monomorphic 0-21.42 %, and no amplification bands 7.14-50% with fragment sizes of 250-2250 kb. The dendrogram of 14 avocado accessions that were tested based on avocado DNA fragments which showed genetic similarity coefficient 26% to

93% or genetic differences coefficient up to 74%

(Fig. 5). The clustering of similarity characters can be divided into 4 groups. Group I consisted of 10 accessions, i.e: Cipedak, E10-I-201, E10-I-204, E10-I-209, E10-II-211, E10-II-213, E10-II-218, E10-IV-268, E10-IV-279, and E10-V-294. Group II contained 2 accessions, i.e: E10-II-219 and E10- III-260. Group III and group IV each consisted of 1 accession, namely: E10-I-206 and E10-II-215, respectively.

Fig. 5. Dendogram based on DNA fragmen of avocado leaves

Table 9. Percentage of polymorphic and size of DNA-RAPD fragments of 13 M1 accessions and 1 Cipedak avocado accession

No. Primer Polymorphic (%) Monomorphic (%) No amplification (%) Fragment size (bp) Lower Upper

1 OPA-03 78.57 0.00 21.42 500 1250

2 OPA-13 85.71 7.14 7.14 300 1500

3 OPC-14 57.14 14.28 28.57 625 1250

4 OPG-01 50.00 0.00 50.00 300 2000

5 OPX-15 71.42 14.28 14.28 400 1500

6 OPY-15 71.42 7.14 21.42 250 1000

7 RAPD-5 50.00 0.00 50.00 500 2000

8 UBC-811 85.71 7.14 7.14 500 1250

9 UBC-817 71.42 7.14 21.42 500 1500

10 UBC-820 50.00 21.42 28.57 300 2250

11 UBC-826 92.85 0.00 7.14 500 1500

12 UBC-834 92.85 0.00 7.14 300 1250

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Twelve primers with polymorphic amplification of OPA-03, OPA-13, OPC-14, OPG-01, OPX-15, OPY-15, RAPD-5, UBC-811, UBC-817, UBC-820, UBC-826, and UBC-834 were used in DNA-RAPD analysis in avocado. Azizah et al. (2019) said the results of DNA bands in PCR amplification were greatly influenced by the quality of the DNA. It is known that too low concentration and purity of DNA results in unclear DNA bands on the electrophoretic gel mold. Priyadarshan (2019) stated that the amplification of the resulting primer polymorphism depends on how much the primer recognizes its homolog. The more DNA fragments attached to the primer, the more the number of fragment bands produced. Testing of 13 M1 accessions of avocado and one avocado cv. Cipedak using 12 primers resulted in various polymorphisms and fragment sizes. McGregor et al. (2000) stated that the amplification of polymorphism is a visualization of the diversity of DNA fragments from the plant being tested. Differences in the number and pattern of fragments indicate differences in DNA sequences so that they can be used as an assessment of genetic diversity.

Examination on 14 avocado accessions based on avocado DNA fragments were resulted a dendogram which genetic similarity coefficient 26% to 93% or genetic differentiation coefficient up to 74% (Fig. 5). The coefficient among avocado accessions is a similarity value that can form genetic groups and link among genetic groups to form tree and branching patterns. Martono & Syafaruddin, (2018) stated that a high similarity coefficient value indicates close or uniform kinship. On the other hand, a low similarity coefficient value indicates distant kinship or genetic diversity with high diversity.

There were 4 different accession groups on the dendogram of 14 avocado accessions based on macro-morphological, micro-morphological and DNA-RAPD characters. The accessions that were consistent within their groups from the 3 dendrograms were group I: Cipedak, E10-I-209, E10-II-211, E10-II-213, and group II: E10-II-219.

There were no consistent accessions from groups III and IV. The occurrence of this phenomenon is suspected because there is a possibility that the macro-morphological and micro-morphological characters are part of the DNA-RAPD marker.

Another possibility is that the DNA-RAPD marker obtained is not a DNA marker that is the morphological character observed. In addition, there

is also the possibility that macro-morphological characters may not necessarily correlate with micro- morphological characters.

This study informed that the tested accessions were given various genetic similarities and indicated the induction of genetic mutations.

Tounekti et al. (2017) stated that mutations occur due to changes in the DNA base chain, causing changes in phenotypic or genotypic properties.

CONCLUSION

Gamma irradiation on Cipedak avocado shoots with a dose of 10 Gy was able to significantly change the macro morphological characters (periole length, blade length, and blade width), micro morphology (thrichome density and thrichome length), and genetic properties of the leaves as determined by changes in the number of bands amplification results. The coefficient of difference in macro and micromorphological characters was 33% and 28%, respectively, while for DNA-RAPD analysis the difference was 74%. Plant maintenance and character observation until the plants bear fruit need to be done to determine the level of change in fruit characters.

ACKNOWLEDGEMENT

Author thanks to Khoirul Mukminin, B.Sc as Chief of Instalation for Agricultural Technology Research and Assessment (IP2TP) Cukurgondang – Indonesian Tropical Fruits Research Institute (ITFRI) who provide seed nursery facilities for conducting research. Author thanks also to Dr.

Raden Heru Praptana as an ITFRI director who provide assistance of seed laboratory facilities for DNA-RAPD analysis.

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