An Effort of Mutation Breeding by Oryzalin and Gamma Rays on Wild Raspberry ( Rubus sp . ) in Cibodas Botanical Garden

Currently, Cibodas Botanical Garden (CBG) has a domestication programme of wild raspberries (Rubus sp.) from Indonesia mountain forest. One of the activities in the domestication programme is fruit breeding. In order to improve the quality of wild raspberry, two mutagens i.e. chemical (oryzalin) and physical (gamma rays) mutagens were used in the breeding programme. Moreover, seed of wild raspberries from the collection of CBG were used in the programme of mutation breeding. The results showed that each species of wild raspberries has different response on the mutagen. Percentage of seed germination were affected by mutagen of oryzalin and gamma rays. Rubus fraxinifolius and Rubus rosifolius seeds were still able to germinate after treated by 100 μM of oryzalin. In the other hand, with increase in radiation above 300 Gy a reduction in percentage of germination was observed in M1 generation of Rubus lineatus and Rubus chrysophyllus.

berry as a fruit crop becomes a CBG programme.An improvement of fruit quality was conducted by three steps, i.e. selection, mutation breeding, and cultivation.At present, we are using four species of Rubus (R. fraxinifolius, R. rosifolius, R. chrysophyllus and R. lineatus) in the fruit breeding, selection, and cultivation programmes.
Mutation is an approach for species improvement and development.Several mutagen were used in the mutation breeding programme, such as physical and chemical mutagens (Medina et al., 2004), to improve and develop a Rubus species.In order to domesticate and develop wild raspberries from Indonesian Mountain Forests, this activity is aimed to improve the genetic variance of wild raspberry for cultivation purposes through mutation breeding.

METHODS
The research was conducted in the laboratory and greenhouse of Cibodas Botanical Garden-Indonesian Institute of Sciences (LIPI), West Java, in 2014-2015.Plant material were used in this research are seeds from four species of wild raspberries of CBG collection i.e.R. rosifolius, R. chrysophyllus, R. fraxinifolius and R. lineatus.Seeds were obtained from mature fruit.Induction of mutation Rubus spp. was divided into two different types of mutagens, i.e. oryzalin and gamma rays.
Application of gamma rays irradiation of Rubus spp.had been conducted at the Center for the Application of Isotopes and Radiation Technology, BATAN.Gamma rays with six doses (100 Gy, 200 Gy, 300 Gy, 400 Gy, 500 Gy, and control) were subjected to the seed of R. lineatus, while seeds of R. chrysophyllus used eleven doses of gamma ray (50 Gy,100 Gy,150 Gy,200 Gy,250 Gy,300 Gy,350 Gy,400 Gy,450 Gy,500 Gy) Rubus spp's.seeds were germinated on wet tissue paper in a petridish.Percentage of germina-

INTRODUCTION
Cibodas Botanical Garden (CBG) or Bergtuin te Tjibodas, part of Indonesian Institute of Sciences, was established on April 11 th , 1852 (Suryana & Widyatmoko, 2013).Cibodas Botanical Garden, an institution for ex situ plant conservation, provides breeders with the basic resources, i.e. genetic variation from wild species for breeding purposes.Based on International Agenda for Botanic Gardens in Conservation (2000), some of major activities of botanic gardens include; conservation biology, environmental education programmes, ethnobiology research, herbarium studies and plant taxonomy, horticultural research and training, laboratory research including in vitro plant cultivation, seed store and tissue banking, tourism, and many more.
Botanical garden has an important role in crop improvement.Moreover, in the domestication process, the useful species were chosen and cultivated, and improved by continuous selection.Through that process, the genetic improvement of those species have been achieved by selection, first from natural seedling population and then from the field that continued unique genotype fixed by vegetative propagation (Lestari & Surya, 2016).Since 2014, wild Rubus domestication programme was ongoing projects in CBG.This project was focusing on utilizing and developing wild Rubus from Indonesian mountain forests.
Plant breeding is a deliberate effort by humans to nudge nature, with respect to the heredity of plants, to an advantage.The changes made in plants are permanent and heritable (Acquaah, 2012).Plant breeding is the purposeful manipulation of plant species in order to create desired genotypes and phenotypes for specific purposes.Plant breeding can be accomplished through many different techniques ranging from simply selecting plants with desirable characteristics for propagation, to more complex molecular techniques.Several techniques such as hybridization, mutation, selection, molecular marker were applied in the plant breeding programme.
Statistical analysis was used to analyze the data.Referring to the procedure of data analysis from Gomez & Gomez (1995), all the data were expressed as the mean.

RESULTS AND DISCUSSION
Mutation breeding on Rubus fraxinifolius Chemical mutagen, oryzalin, was subjected to the seed of R. fraxinifolius.Five combinations of oryzalin concentration and submersion time, i.e. 0 µM in 0 hour (control), 50 µM in 6 hour, 50 µM in 24 hour, 100 µM in 6 hour, and 100 µM in 24 hour, were applied to seeds.Seed that had been treated by oryzalin was germinated in a petridish on wet tissue paper.Moreover, Figure 1 shows that germination percentage and survival of germination of R. fraxinifolius seed treated by oryzalin was higher than control.The highest of germination percentage was showed by the combination of 24 hours of submersion with 50 µM of oryzalin (75.76 %) and 6 hours of submersion with 100 µM of oryzalin (75.76 %).It showed that the higher levels of oryzalin concentration and the longer of submersion time were related to a germination percentage of R. fraxinifolius seed.In the other hands, the percentage of survival germination after transplanting was showed by the combination of 6 hours of submersion with 50 µM of oryzalin (92.59 %) and 24 hours of submersion with 100 µM of oryzalin (92.59 %).Furthermore, Figure 2 shows the variation of leaf morphology in R. fraxinifoilus treated by oryzalin.Physical mutagen, gamma ray with six doses (0, 100, 200, 300, 400, 500), were subjected to the seed of R. lineatus.The result shows that the seeds of R. lineatus on doses 0, 100, 200 and 400 gray were still able to germinate.The highest percentage germination was showed in doses 100 gray (Figure 3).

Mutation breeding on Rubus lineatus
Physical mutagen, gamma ray with eleven doses (0,50,100,150,200,250,300,350,400,450,500), were subjected to the seed of R. chryso-Figure 2. The leaf morphology variation of R. fraxinifoilus after treated by oryzalin phyllus.Except on the doses of 500 gray, germination percentage increased after treated by gamma ray.The highest of germination percentage was showed on 50 gray (Figure 4).Chemical mutagen, oryzalin with eleven doses (0,10,20,30,40,50,60,70,80,90,100), were subjected to the seeds of R. rosifolius.Three levels of time (1, 6, 24 hours) were used to submersion the seed of R. rosifolius.The results showed that the highest of germination percentage was 98%, while the seeds of R. rosifolius was soaked 24 hours in 30 µM of oryzalin (Table 1).Moreover, after a year growth in a petridish, all germinated seeds were transplanted onto the pot in the plastic house.Base on survival percentage of R. rosifolius mutants, Table 2 shows that on 14 days after transplanting, mutants treated by higher concentration of oryzalin is more adap-table.The lowest survival percentage was mutant of R. rosifolius treated in 10 µM of oryzalin.
Oryzalin and gamma rays affected the seed germination of R. fraxinifolius, R. lineatus, R. chrysophyllus, and R. rosifolius.The results showed that the increasing concentration of oryzalin was able to rise up the percentage germination of R. fraxinifolius (Figure 1) and R. rosifolius on 6 and 24 hours of submersion time (Tabel 2).The results seems unusual because generally the percentage of germination decreased as the concentration of mutagens increased.The reduction in germination may be due to genetic and physiological proses inhibited by the mutagens resulting in cell maturity (Umavathi & Mullainathan, 2014).On the other side, Roychowdhury & Tah (2011) reported that increasing the doses of colchicine were able to increase the germination percentage of Dianthus caryphyllus.Their result was in line with our experiment, which is the increasing concentration of oryzalin was able to increase the percentage germination of R. fraxinifolius and R. rosifolius.Furthermore, the range of gamma rays doses tested did not significantly affect the germination percentage of R. lineatus and R. chrysophyllus seeds.It is slightly different with certain studies performed on seeds of several species such as Withania samnifera (Bharathi et al., 2013), Citrus jambhiri (Sharma et al., 2013), Cajanus cajan (Ariraman et al., 2014) that a gradual decrease in seed germination from lower doses to higher doses of gamma rays were observed.In the other hand, Giovani et al. (2015) reported that the effect of gamma rays at 50, 100 and 200 Gy were able to increase seed germination of Rosa hybrida.Their results are in line with our experiment, which was the seed germination of R. lineatus increase in 50,100,150,200,250,300,400,450 Gy and R. chrysophyllus in 100 Gy.
Mutation induction is a part of domestication programme, in order to utilizing and cul-  (Umavathi & Mullainathan, 2014).However, the percentage of seed germination was not always decreased with increasing doses/concentration of gamma rays and oryzalin in wild raspberries.In general, these results show that the mutagens have affected on seed germination.Moreover, small doses indicated that the mutagens have exerted a stimulus effect on seed germination, but in some cases the increasing doses have exerted an inhibitor effect.Furthermore, oryzalin and gamma rays were able to affect the seed germination of wild raspberries.Mutagenic agents, such as oryzalin and gamma rays, with certain doses may be used to enhance seed vigor and increase the percentage of germination of wild raspberries

CONCLUTION
A preliminary study of mutation breeding is an effort to develop wild raspberry in Cibodas Botanical Garden.Both of mutagens, oryzalin and gamma rays affectedthe germination of Rubus sp.Moreover, less than 100 µM of oryzalin with 6 hours submersion time and less than 60 µM of oryzalin with 24 hours submersion time were the recommendation to treat the Rubus's seeds by oryzalin mutagen.In the other hand, the effective doses of gamma rays for Rubus's seeds were less than 300 Gy.

Figure 1 .
Figure 1.The effect of submersion time and doses of oryzalin on the germination percentage of Rubus fraxinifolius Mutation breeding on Rubus lineatus.

Figure 3 .Figure 4 .
Figure 3.The effect of gamma ray irradiation on the germination percentage of Rubus lineatus

Table 1 .
The effect of submersion time and doses of oryzalin on the percentage of Rubus rosifolius germination

Table 2 .
Survival percentage of R. rosifolius mutants on 14 days after transplanting.