INDUCED IN VIVO MUTAGENESIS USING COLCHICINE IN HYDROGEN PEROXIDE ON M2 COWPEA, YARDLONG BEAN AND COMMON BEAN

Research on doubling plant chromosome using colchicine has been carried out for years with water as its solvent. In this study, an innovation was carried out by trying to do colchicine treatment in hydrogen peroxide. In this in vivo induced mutagenesis research, colchicine in hydrogen peroxide was administered as mutagen on seeds three species of crops i.e. mutan M2 cowpea, yardlong bean, and common bean by soaking method. The results of this research showed that three times multistep mutant (M3) of cowpea was having mutant characteristics that the shape of its leaflet changed from rhombus to ovate. Besides, both yardlong bean varieties used in this research (BLR and KTR) showed indication of a mutant crop that having compound leaves with more than three leaflets in a stalk. Common bean of PTW variety was not attacked by any aphid but it was attacked by leafminer. Finally, we found “rainbow common bean” from this research.


INTRODUCTION
Cowpea or blackeyed bean is named "Tunggak" bean or "Tolo" bean in Indonesia.
This kind of bean has been consumed since the ancient time and it is still cultivated until at present besides also planted as a cover crop.
Yardlong bean and common bean are in great demand in Indonesia and used to be eaten as salad or it cooked as a mixed vegetable dish.
Colchicine has been used to induced mutagenesis to produce mutant crop through chromosome multiplication resulting in larger both vegetative and generative plant organs. Dermen and Emsweller, 1961 used colchicine as chromosome multiplication agent for polyploidization process of plants with concentration ranged 0.1-1% with water solvent or 10% water and glycerin mixture. In medicine, colchicine is used as an anti-inflammatory drug for the treatment of tophus (Dalbeth et al., 2014), Mediterranean fever and cardiovascular disease (Campbell et al. 2015). Results of our previous research showed that the effect of colchicine in hydrogen peroxide on cowpea were 1) temporary leaf change, 2) pod color change from green to purple (Susrama and Wirawan, 2017), 3) change of leaflets number per leaf stalk from three leaflets to four and five leaflets, and 4) gigas mutant cowpea, which having larger phenotype (Susrama and Wirawan, 2018).

METHODOLOGY
All seeds of mutant M3 cowpea, yardlong bean and common bean were selected visually that showing no pest damage, uniform in size and uniform in color.
This research divided into three packages.
The first package was induced mutagenesis on cowpea consisted of 1) 25 seeds of twice multistep mutant cowpea (M2) treated with 0.01 percent colchicine in 3 percent hydrogen peroxide, 2) untreated 25 brown color cowpea seeds that free from cowpea mosaic virus (CPMV) incidence produced from our previous research and it tried as candidate of CPMV cowpea resistance (Susrama and Wirawan, 2018), and 3) 25 M2 cowpea seeds soaked in 3 percent hydrogen peroxide as control. All those seeds after 72 hours were then planted in Taman  All seed categories then planted. Observation variables were leaf morphological and structural changes, pest and disease incidence, color and size of pods, and seed weight.

Induced mutagenesis on cowpea
The three times multistep mutant (M3) cowpea and the cowpea variety as resistance candidate against CPMV were still showing mutant characteristic such as leaf-shaped changing from rhombus to ovate and its compound leaves having four or five leaflets, indicating that the nature of mutant is still present, means that those traits were inherited.   Table 1). This result showed that multi-step colchicine effect was decreased, in other words, the effect of colchicine was better on origin variety.   (Table 3). This result showed that colchicine in 3% hydrogen peroxide did not increase the seed weight of cowpea seeds significantly.  showed that no colchicine effect was shown on pods length of KTR yardlong bean (Table   5).

Aphid incidence on yardlong bean
The attack of aphid pests (Fig. 1)  attacking yardlong bean KTR variety (508 aphids). The number of BLR variety individuals attacked (14 individuals) was also four times lower than KTR variety (59 individuals). Based on the data of incidence time and aphid abundance, it was obvious that yardlong bean BLR variety has a higher resistance against aphid. Colchicine multistep treated yardlong bean KTR variety was afflicted by 269 aphids, almost two times less than on control (508 aphids). The number of crops attacked was also less, it was 37 percent lower than the number of aphids on control.
Four pods were attacked among twenty pods observed or in the percentage of twenty.
Surprisingly, fire ant did not occur both in control and colchicine-treated yardlong bean KTR variety. As far as we know, up to now, fire ant was not an insect pest on any crop.
Although it was not something common, however, the visual evidence of this research results indicated that fire ant incidence occurred on yardlong bean pods.   Due to colchicine solution treatment solution on PTW common bean variety, we found that six percent or three crops out of twenty sample crops produced pods with a different color i.e. light green pod with purple spots (Fig. 4). Such kind of bean is called colored common bean pod or rainbow common bean pod. As far as we know, this kind of result was never published in any scientific journal yet and at present, the rainbow common bean is not available in the Indonesian market.