Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(1): 15-27, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1202 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Rajani Singh, Girjesh Kumar (2022) Analyzing frequency and spec- trum of chlorophyll mutation induced through Gamma ray and Combination treatment (Gamma + EMS) on genetic paradigm of Artemisia annua L.. Cary- ologia 75(1): 15-27. doi: 10.36253/caryo- logia-1202 Received: January 31, 2021 Accepted: March 23, 2022 Published: July 6, 2022 Copyright: © 2022 Rajani Singh, Girjesh Kumar. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID RS: 0000-0001-9782-6754 Analyzing frequency and spectrum of chlorophyll mutation induced through Gamma ray and Combination treatment (Gamma + EMS) on genetic paradigm of Artemisia annua L. Rajani Singh*, Girjesh Kumar Plant Genetics Laboratory, Department of Botany, University of Allahabad, India *Corresponding author. E-mail: singh.rajani1995@gmail.com Abstract. For the development of genetic programs with novel characteristics induced mutagenesis has been used extensively. Chlorophyll (chl) mutations are considered as the most dependable indices for assessing the efficiency of different mutagens in induc- ing the genetic variability in crop plants and are also used as genetic markers in basic and applied research. In the present scenario of high health susceptibility, the global demand for natural medicine derived from plant species has increased enormously. Sweet wormwood (Artemisia annua Linnaeus) – an important medicinal plant spe- cies with immense remedial values, was selected for the present study and exposed to gamma rays at 100 Gy, 200 Gy, 300 Gy and combination treatments with 100 Gy + 0.1%EMS, 200 Gy + 0.1% EMS, 300 Gy + 0.1%EMS. Meiotic study was also done and various cytological aberrations were observed in M2 generation like stickiness, preco- cious, scattering, laggard and bridge etc. The frequency of induced chl mutation var- ied in different mutagen treatments. Eight different types of chl mutants namely albina, chlorina, xantha, aurea, viridis, yellow viridis and tigrina etc. were recorded in M2 gen- eration on plant population basis. The frequency of xantha mutants was quite high in both the treatments but in gamma exposed set it was followed by albina whereas in combination treatments viridis was second highest mutant. In different mutants quan- titative analysis of chl pigments was also done and content was highest in viridis i.e. 3.86µg/ml FW and lowest in albina i.e. 0 µg/ml FW . Although chlorophyll mutations thought to be lethal in nature, but present study has proven to be a milestone in iden- tifying the threshold dose of a mutagen that would increase the genetic variability and induces new trait in Artemisia annua Linnaeus. Keywords: Artemisia annua L. (Linnaeus), chlorophyll mutants, cytological anoma- lies, gamma rays, EMS. INTRODUCTION The medicinal plant Artemisia annua, also known as Sweet Wormwood or Sweet Annie, is one of the top 10 pharmaceutical crops which are get- ting intensive worldwide scientific consideration as this valuable treasure is the only source for the commercial pharmaceutical production of the ses- 16 Girjesh Kumar, Rajani Singh quiterpene lactone artemisinin (Prasad and Das 1980). Artemisia has been applied in the traditional medi- cine, for the treatment of diabetes, depression, insom- nia and stress, to clear the lymphatic system and in the oncotherapy. The whole plant of A. annua L.is still the most economic source of artemisinin, and the develop- ments of high-producing plants of A. annua L. appear to be the main direction to obtain large quantities of relatively inexpensive artemisinin. For any success- ful crop improvement programme, genetic variability plays an important role because it provides a spectrum of variants for effective and better selection which can be obtained using mutation, hybridization, recombi- nation and selection processes (Dhumal and Bolbhat 2012). Mutational breeding involves high energy radia- tion such as X, β and γ-rays, which are electromag- netic radiations that initiate or inhibit the growth and differentiation of plant cells and organs (Hasbullah et al. 2012) they could also modify physiological charac- teristics of plant to create new mutants for production of high amounts of commercially important metabo- lites. Ionizing radiation has been recognized as a pow- erful technique for plant improvement of medicinal plants (Vardhan and Shukla 2017). This technique cre- ates genetic variability in plants, which can be screened for desirable characteristics. Previously, Koobokurd et al. (2008) reported a method for establishing in vitro plantlet variants of A. annua using low-dose gamma irradiation. By using gamma rays many high yielding mutant varieties have been developed world wide, which are resistant to biotic and abiotic stresses with improved quality (IAEA 2017). The success of mutation breed- ing programme largely depends on selection of prom- ising mutants based on phenotypic characters (Arisha et al. 2015). EMS, as a chemical mutagen, can be used as a supplementary approach to improve desired iden- tifiable characters such as yield related characters (Bot- ticella et al. 2011). Chemical mutagens are not only mutagenic themselves but also affect mutation in spe- cific ways when combined with radiation (Reddy and Smith 1981). It produces random point mutations in genetic material. So, mutation frequency, detected using various techniques, displays a wide range of variation in combination treatment where plants seeds exposed to physical mutagen followed by chemical mutagen. During M1 generation, probably identification of reces- sive character is difficult only mutations of dominant characters can be identified. In the M2 generation, the mutation will segregate to create homozygotes for reces- sive or dominant alleles (Page and Grossniklaus 2002). The most effective way to identify the phenotypic muta- tion is Visual screening which can be used as a primary indicator to select plants that have desired characters, for example: disease resistance, f lowering earliness, plant height or growth period (Østergaard and Yanofsky 2004). Gene mutations influencing the green coloration of photosynthetically active parts are among the most common spontaneous or induced alterations arising in higher plants (Kolar et al. 2011). Although chlorophyll mutations are generally not useful for plant breeding purpose because of not having any economic value due to their lethal nature, their study could be useful in identifying the suitable mutagen and threshold dose of mutagen that would increase the genetic variability and number of economically useful mutations in the segre- gating generations (Wani and Anis 2004). The chloro- phyll mutation frequency is an indicator to predict the frequency of factor mutations and thus an index for evaluation of genetic effects of mutagens (Walles 1973). In addition, chl mutations are important for identify- ing gene function and elucidation of chl metabolism and its regulation15. The occurrence of chl mutations after treatments with physical and chemical mutagens have been reported in several crops (Swaminathan et al. 1962; Sharma and Sharma 1981; Reddy and Gupta 1989; Mitra 1996; Kharkwal 1998; Solanki 2005; Wu et al. 2007). Induced mutations can rapidly create vari- ability in quantitatively and qualitatively inherited traits in crops. Genetic variability has been induced through mutagenesis in several plants, but the information avail- able in A. annua L. is meager. In the present study attempt has been made to understand the compara- tive response of physical and chemical mutagens on A. annua, with a view to determine the mutagen and treat- ment causing maximum chl mutations in M2 genera- tion and also on cytological parameter. MATERIALS AND METHODS Plant materials M2 seeds generated from the M1 generation of vari- ety EC-415012, were used in this study. The M1 seeds were produced by exposing separate 1000 dry seed samples (for each dose) to 100Gy, 200Gy and 300Gy at a dose rate of 15.48 Gy/min of gamma radiation using a 60Co (Cobalt 60) gamma source under ambient con- ditions at the National Botanical Research Institute (NBRI), Lucknow and for combination treatment con- centration of Ethyl Methyl Sulphonate (EMS) solu- tion of 0.1% was prepared. EMS solution was settled in a 0.1M phosphate buffer at pH 7.0 to avoid rapid hydrolysis (Bosland 2002). Gamma ray treated (100Gy, 200Gy, 300Gy) seeds were presoaked in water for 6h 17Analyzing frequency and spectrum of chlorophyll mutation induced through Gamma ray and Combination treatment then treated with the above-mentioned concentration of EMS at 20°C with orbital shaking(110rpm) along with control (untreated) seeds. Seeds were then thor- oughly washed under running water then transferred to Petridishes containing wet filter paper and kept in a growth chamber at 25°C in the seed germinator for germination (at 2 days after the treatment).Control seeds were exposed to the same conditions except for the EMS treatment. Experimental plan and procedure The experiments were carried out in the first week of the month january at Roxburg Botanical Garden, Department of Botany. The M1 plants are individually harvested and sown as M2 families. according to the Pedigree Method; the M1 plants are individually harvest- ed and plants with probable mutants following pheno- typic observations as plant habit variation in leaves (chl mutants), early plant vigour (poor, good and very good), plant height (short stature, up to top of the plant), sown as M2 families .Sweet wormwood M2 lines were grown in the field (geographical location is 25o27’43.01”N, 81o51’10.42”E) in randomized complete block design (RCBD) and allowed to produce the M2 seeds.. The net plot size was 4 m _ 4 m, with nine rows (each 4 m long) with a 45 cm distance between two rows and approxi- mately 20 cm distance between two plants. The untreat- ed seeds (control) were planted in the first row of each plot. For weed control plots were irrigated during vegeta- tive growth and the plants were harvested individually at full maturity. Germination (%) taken after 7 days and plant survival (%) was recorded after 14 days for each mutagenic treatment as well as control in M2 generation. After a month Six phenotypic traits were analysed and recorded as plant height (cm), internodal length (cm), leaf area(per m2), No. of primary branches, days to 50% flowering and days to maturity etc. Cytological investigations For the cytological analysis young floral capitula of control and variant plant of Artemisia annua L. with appropriate size were fixed in Carnoy’s fixative (Alcohol 3: Glacial Acetic Acid 1) for 24 hrs and then transferred in 90% alcohol to preserve the capitula for meiotic study. Anthers were teased and stained in 2% acetocarmine, followed by squash preparation. Slides were observed under the microscope and pollen fertility was evalu- ated by acetocarmine stainability test. The snapshots of chromosomes were captured by the help Pinnacle PCTV software. For pollen fertility, mature capitula having pollen grains were dusted over glass slide and stained with acetocarmine and mounted with glycerine. Then observed under optical microscope to count the frequen- cy of fertile and sterile pollen grains. Pollen fertility (%) = No. of fertile pollen × 100 Total no. of pollen Quantification of Photosynthetic pigments Photosynthetic pigment was quantified according to Lichtanthelar and Welburn (1983)method. 20mg of leaves were taken and dissolved in 5ml of 80%acetone. Solution was extracted and were centrifuged at 15000 rpm for 10min at 10°C. The supernatant volume was diluted with 80% acetone. O.D. was taken at three dif- ferent wavelength i.e. 470nm, 663nm and 646nm in the spectrophotometer and finally chl a, chl b calculated. Observations recorded and statistical procedure The M2 generation was screened for phenotypic variations from germination to harvesting. The fre- quency of the mutant plants out of the total number of individuals in M2 generation was calculated. The muta- genic frequency was estimated as the percentage of seg- regating M1 plant progenies. Chl mutations were clas- sified into various types based on the method followed by Gustafsson(1940). For statistical analysis in the table, three replicates for each treatment were used. Statistical analysis was performed using the SPSS 16.0 software. A oneway analysis of variance (ANOVA) and Duncan’s Multiple Range Test (DMRT, P < 0.05) was conduct- ed for mean separation and the graph was plotted by using sigma plot 10.0 software. Actual mean and stand- ard error were calculated and the data was subjected to analysis of variance. RESULTS Germination and Survival percentage Fig. 1 shows that germination percentage and plant survival were significantly declined as the mutagen- concentration increased. Conspicuous variations were recorded after both the treatment in sweetworm wood. Athigher doses germination and survival percentage was found to be 76.60%, 61.76% (in Gamma ray) and 33.83%,24.66% (in Gamma+ EMS) respectively. 18 Girjesh Kumar, Rajani Singh Chl leaf variations In the present appraisal, the frequency of chl mutants calculated as percent of M1 plant progenies and M2 plants basis were presented in Table 1. It was observed that the frequency of induced chl mutant was increased with an increase in the dose of gamma ray and combination treatment but at the maximum doses mutation frequency was decreased. At the 300Gy muta- tion frequency was recorded as 3.60%(in M1 generation) and 0.98%(in M2 generation)in gamma ray treatment while it was 2.60%(in M1 generation) and 0.79% (in M2 generation) in combination treatment. It was observed that the mutation frequency in M2 generation was more in combination treatment of 100Gy+0.1% EMS( 2.82%) than gamma ray alone (1.09 %) M2 generation depicts presence of broad chl mutantspectrum, comprising total 8 type mutants. The spectrum of M2 chl mutants includ- ed albino, xantha, chlorina,maculata, Tigrina, auria and viridis are presented in Table 2. In both the treatment, frequency of xantha mutants (in Fig. 2) was maximum (all total) 92.75% (in gamma) and 103.78% (in combined treatment), followed by viridis, albino and yellow viridis in gamma treatment while in gamma+EMS, it was fol- lowed by albino, maculata and chlorina. The least fre- quency of auria type of chl Mutant was recorded 28.14% (in gamma) and 11.11% (in combined treatment). Albina (Fig. 4 e & g) mutants were completely lack of chl and could survive only a few days. Chlorina (Fig. 4 a,b,c) and yellow viridis ( Fig. 4 h & i) had green and yellow green leaves, respectively, are lethal mutations. Aurea (Fig. 3d) had golden yellow coloured leaves and xantha (Fig. 3b) had pale yellow coloured seedlings, tig- rina (Fig. 4d) had yellow colour at edges of the leaves furthermore viridis (Fig. 3c ) had dark green colour, these type of mutants not only survive but they complete its full lifecycle. Chl content in chl deficient mutants The concentration of green pigmentation in the leaves differed among the different types of chl defi- cient mutants, ranging from chlorina to xantha type of mutants. The chl content (Fig. 5) of various mutants and along with control tissues were examined, the mutants contained significantly less chl than normal plants accept viridis mutant. Among the mutants, albi- na type was totally devoid of chl while xantha (0.83µg g-1FW) and aurea (0.96µg g-1FW) contained the least Figure 1. Effect of Gamma ray and Combination treatment on ger- mination and survival in Artemisia annua L. (M1 generation). Table 1. Frequency of chlorophyll mutants induced through gamma and combination treatment (Gamma+EMS) in M2 generation of Arte- misia annua L. Treatments No. of M1 plant progeny M1 segregates for mutation M2 plant scored M2 mutants Mutation frequency (%) M1 Plants frequency M2 plant frequency Gamma rays (Gy) Control 500 1090 100 500 27 1097 12 5.40 1.09 200 500 21 1070 23 4.20 2.15 300 500 18 921 9 3.60 0.98 Combination treatment (Gy+ %) Control 500 1073 100+0.1% 500 30 1038 28 6.00 2.82 200+0.1% 500 24 994 13 4.80 1.31 300+0.1% 500 13 882 7 2.60 0.79 19Analyzing frequency and spectrum of chlorophyll mutation induced through Gamma ray and Combination treatment chl and those of viridis (3.86µg g-1FW) the most. Th e other chl mutants maculata (1.24µg g-1FW), tigrina (1.06µg g-1FW), chlorina (2.46 µg g-1FW) and yellow vir- idis (3.01µg g-1FW) contained signifi cantly less chl than those of control (3.24 µg g-1FW). Meiotic study Meiotic study was done in chl mutant plants which were identifi ed in M2 generation to screen out genetic disturbance caused by both the mutagen. In control plants 9:9 standard configuration was found. Differ- ent types of anomalies were exhibited by treated sets i.e. precocious movement, stickiness, scattering, laggards, bridges, disturb polarity etc. (Fig. 6D–L). Stickiness and multivalent was found to be predominant abnormality in treated sets. Table 3 represented various abnormality frequency and Total Abnormality(TAB) percentage in treated sets. At lower dose of both the treatment TAB% was found to be 6.24±0.15% (Gamma) and 8.93±0.17% (Combined treatment) while at higher dose of both the treatments TAB% was shoot up to 19.71±0.22% (Gam- ma) and 26.63±0.48% (Combined treatment) . It shows that abnormality percentage is dose dependent. The results shown in Table 4 indicated that the growth habit of all mutant plants (plant height, inter- Table 2. Spectrum and Frequency of chlorophyll mutant in diff erent mutagenic treatments. Treatment Total chl mutants in M2 generation Th e relative percentage of chlorophyll mutants(%) Albina Xantha Chlorina Tigrina Maculata Viridis Yellow Auria Gamma ray (Gy) 100 12 16.67 33.33 8.33 8.33 0 8.33 16.67 8.33 200 23 13.04 26.09 8.70 13.04 8.70 13.04 8.70 8.70 300 9 11.11 33.33 0 0 0 22.22 11.11 22.22 Gamma+EMS 100+0.1% 18 11.11 44.44 16.67 0 0 0 16.66 11.11 200+0.1% 13 15.38 30.78 15.38 15.38 0 15.38 15.38 0 3000+0.1% 7 14.29 28.57 0 14.29 28.57 14.29 0 0 0 20 40 60 80 100 120 Alb ina Xa nth a Ch lor ina Tig rin a M ac ula ta Vir idi s Ye llo w vir idi s Au ria gamma gamma+EMS Figure 2. Chlorophyll mutations frequency on mutagen basis in Artemisia annua L. Figure 3. Chlorophyll mutants in Artemisia annua L.: a- Control, b-Semi-xantha mutant at seedling stage, c- Viridis mutant, d- aurea mutant 20 Girjesh Kumar, Rajani Singh nodal length, leaf area, No. of primary branches, days to 50% f lowering and Days to maturity) were dif- ferent than those of the control plants. As height (cm) of the control plant was observed 103.20±0.88 although at 100Gy and 100±0.1% it was increased i.e. 112.30±1.01and 110.30±1.09 which decreased as the Figure 4. Different types of chlorophyll mutants in Artemisia annua L. a. b. c. Chlorina mutant, d. Tigrina mutant, e. g.. Albina mutant, f. maculate mutant, h. i. Yellow viridis mutant 21Analyzing frequency and spectrum of chlorophyll mutation induced through Gamma ray and Combination treatment doses of mutagen increases. Internodal length, leaf area and primary number of branches were improved at the lower dose of gamma rays but as the doses increases all these character significantly minimally decreased. At lower dose of gamma and gamma+EMS plant flowers earlier and matures faster as compared to control. Con- trol plants were flower in 56 days and mature in about 155-156 days. While At 100Gy and 100+0.1%, days to 50% flowering was observed 50.00±0.57 and 52.00±1.03 furthermore days to maturity was 145.00±4.50 and 143.00±1.00. Fig. 7 shows the dwarf variant and tall variant. Dwarf variant was found at 300+0.1% while tall variant screen out at 100Gy dose and also some plant become prostrate as shown in Fig. 7A. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 Control Chlorina Maculata Tigrina Viridis Yellow viridis Albina Auria Xantha Figure 5. Chlorophyll content in normal type and chlorophyll defi- cient mutants of Artemisia annua L. Figure 6. Different types of abnormalities in treated plants of Arte- misia annua L.: A. Stickiness at metaphase I, B. Two precocious chromosome at Sticky metaphase I, C. Stickiness at unoriented Anaphase I, D. One laggard chromosome at Anaphase I, E. Two precocious chromosome at Metaphase II, F. One laggard chromo- some at Anaphase II, G. Mononucleate condiotion, H. Binucleate condition, I. Multipolarity(Scale bar= 10.13µm) Figure 7. Some phenotypic variants in Artemisia annua L. A-Pros- trate variant, B- Dwarf variant C- Tall variant. 22 Girjesh Kumar, Rajani Singh Ta bl e 3. A c om pa ra tiv e ac co un t o f C hr om os om al a no m al ie s in in du ce d th ro ug h G am m a ra y an d co m bi na tio n (g am m a+ EM S) tr ea tm en t i n A rt em is ia a nn ua L . D os es (G y) N o. o f PM C ’s ob se rv ed M et ap ha si c A bn or m al iti es ( % ) (M ea n± S. E. ) A na ph as ic A bn or m al iti es ( % ) (M ea n± S. E. ) O th . (% ) T. A b. ( % ) Po lle n fe rt ili ty (% ) Sc Pm St U n M v Sa Br Lg U n St A sy D p C on tr ol 33 0 - - - - - - - - - - - - - - 94 .4 1± 1. 46 a 10 0 31 0 0. 43 ±0 .1 0 1. 08 ±0 .1 0 0. 65 ±0 .1 9 0. 43 ±0 .1 1 0. 43 ±0 .1 1 0. 32 ±0 .1 9 0. 54 ±0 .1 0 0 0 0. 89 ±0 .1 0 0. 65 ±0 .0 1 0. 32 ±0 .0 4 0. 53 ±0 .1 0 6. 24 ±0 .1 5 92 .3 7± 1. 85 a 20 0 29 0 0. 80 ±0 .3 0 1. 25 ±0 .2 8 0. 92 ±0 .1 3 1. 14 ±0 .2 1 0. 93 ±0 .2 5 0. 56 ±0 .1 2 0. 81 ±0 .1 3 0. 80 ±0 .1 9 1. 04 ±0 .0 2 0. 69 ±0 .0 1 0. 93 ±0 .2 5 0. 69 ±0 .0 1 0. 58 ±0 .1 2 11 .2 5± 0. 14 87 .1 9± 1. 87 b 30 0 29 1 1. 84 ±0 .1 2 2. 19 ±0 .2 7 1. 26 ±0 .0 9 1. 95 ±0 .0 9 1. 37 ±0 .1 8 1. 27 ±0 .1 4 1. 38 ±0 .2 1 1. 37 ±0 .1 0 1. 62 ±0 .3 4 1. 72 ±0 .1 8 1. 14 ±0 .0 8 1. 37 ±0 .1 6 1. 25 ±0 .2 9 19 .7 1± 0. 22 70 .2 5± 1. 59 c C on tr ol 35 8 - - - - - - - - - - - - - - 95 .2 2± 0. 58 a 10 0+ 0. 1% 38 5 0. 61 ±0 .1 0 0. 86 ±0 .0 7 1. 31 ±0 .1 9 0. 69 ±0 .0 7 0. 51 ±0 .1 4 0. 51 ±0 .1 4 0. 61 ±0 .1 0 0. 43 ±0 .2 2 0. 60 ±0 .0 7 0. 79 ±0 .1 7 0. 78 ±0 .1 6 0. 61 ±0 .0 9 0. 61 ±0 .0 9 8. 93 ±0 .1 7 85 .6 7± 1. 15 a 20 0+ 0. 1% 37 0 1. 44 ±0 .0 9 1. 44 ±0 .2 3 1. 29 ±0 .6 6 1. 53 ±0 .2 3 1. 08 ±0 .1 4 1. 08 ±0 .0 4 1. 17 ±0 .1 0 0. 90 ±0 .0 6 1. 28 ±0 .2 8 1. 35 ±0 .1 4 1. 46 ±0 .2 9 1. 07 ±0 .1 2 1. 43 ±0 .1 5 16 .5 4± 0. 85 77 .1 2± 1. 73 b 30 0+ 0. 1% 36 2 1. 39 ±0 .3 2 1. 66 ±0 .1 9 3. 96 ±0 .3 8 1. 66 ±0 .0 4 2. 67 ±0 .1 9 1. 57 ±0 .1 0 1. 56 ±0 .3 3 2. 41 ±0 .3 8 1. 95 ±0 .3 2 1. 11 ±0 .1 8 2. 94 ±0 .1 8 1. 74 ±0 .2 0 2. 01 ±0 .4 7 26 .6 3± 0. 48 61 .2 9± 0. 85 c A bb re vi at io ns : S .E .- S ta nd ar d Er ro r; Sc - Sc at te ri ng ; P m - Pr ec oc io us m ov em en t; St - St ic ki ne ss ; U n- U no ri en ta io n; M v- M ul tiv al en t; Sa - Se co nd ar y as so ci at io ns ; A sy - A sy nc hr on ou s; B r- B ri dg e; L g- L ag ga rd ; D p- D is tu rb ed p ol ar ity ; O th - O th er s; T .A b. – To ta l a bn or m al iti es , M ea ns a re fo llo w ed b y lo w er ca se le tt er is s ta tis tic al ly s ig ni fic an t a t p < 0 .0 5. Ta bl e 4. G am m a ra y an d C om bi na tio n tr ea tm en t ( G am m a+ EM S) o n so m e qu al ita tiv e an d qu an tit at iv e tr ai ts o f c hl or op hy ll m ut an ts s ee dl in g in A rt em is ia a nn ua L . ( M 2 ge ne ra tio n) . M or ph ol og ic al tr ai ts C on tr ol M ea n± S. E. C hl or op hy ll m ut an t l in es 10 0 G y M ea n± S. E. 20 0G y M ea n± S. E. 30 0G y M ea n± S. E. 10 0± 0. 1% M ea n± S. E. 20 0± 0. 1% M ea n± S. E. 30 0± 0. 1% M ea n± S. E. Pl an t h ei gh t ( cm ) 10 3. 20 ±0 .8 8b 11 2. 30 ±1 .0 1a 10 8. 22 ±1 .6 0b 86 .3 0± 1. 48 c 11 0. 30 ±1 .0 9a 88 .3 0± 1. 44 b 52 .5 0± 1. 30 c In te rn od al le ng th ( cm ) 7. 50 ±0 .0 9a b 8. 60 ±0 .1 3a 7. 40 ±0 .1 5b 6. 90 ±0 .1 3c 8. 31 ±0 .1 1a 6. 30 ±0 .1 5b 5. 60 ±0 .1 4c Le af a re a (p er m 2 ) 35 .6 0± 0. 48 b 38 .2 0± 0. 47 a 28 .5 0± 0. 46 c 27 .8 0± 0. 42 c 34 .2 0± 0. 42 b 26 .6 0± 0. 49 b 22 .3 0± 0. 43 c N o. o f p ri m ar y br an ch es 25 .0 0± 0. 86 b 30 .0 0± 1. 08 a 27 .0 0± 0. 86 b 26 ±1 .0 6a 21 ±0 .8 3a b 19 ±0 .7 6b 17 ±0 .6 3c D ay s to 5 0% fl ow er in g 56 .0 0± 0. 43 b 50 .0 0± 0. 57 c 55 .0 0± 0. 60 b 66 .0 0± 2. 16 a 52 .0 0± 1. 03 b 65 .0 0± 2. 60 a 79 .0 0± 2. 79 a D ay s to m at ur ity 15 5. 00 ±1 .0 6b 14 5. 00 ±4 .5 0c 15 0. 00 ±4 .0 1a b 17 0. 00 ±3 .5 5a 14 3. 00 ±1 .0 0c 17 2. 00 ±3 .0 5a 15 9. 00 ±3 .6 8b A bb re vi at io ns : S .E .- S ta nd ar d Er ro r, M ea ns a re fo llo w ed b y lo w er ca se le tt er is s ta tis tic al ly s ig ni fic an t a t p < 0 .0 5 23Analyzing frequency and spectrum of chlorophyll mutation induced through Gamma ray and Combination treatment DISCUSSION In mutation breeding programs, the selection of an effective and efficient mutagen concentration and growth condition is essential to produce a high frequency of desirable mutations(Arisha et al. 2014). Chl mutation frequency in M2 generation is one of the most dependa- ble measures for evaluating the mutagen-induced genetic alternations. The spectrum of chl mutations was found to be dependent on the genetic background of the geno- type. Moreover chl mutation frequency increased with the increase in dose of gamma rays both individually as well as in combination with EMS in all the varieties. In the present investigation the germination per- centage and plant survival were reduced significantly. The reduction in germination may be due to the seeds engrossing the mutagen, which subsequently reaches the meristematic regionof seeds and affects the germ cell (Serrat et al. 2014). Also, a reduction in germination may be because of the damage of cell constituents (Kumar et al. 2013), alteration of enzyme activity or delay or inhibi- tion of physiological and biological processes (Talebi et al. 2012).Reduction in plant survival in treated popula- tion may occur due to various factors such as cytogenet- ic damage and physiological disturbances (Sato and Gaul 1967)and disturbances in balance between inhibitors of growth regulators and promoters (Meherchandani 1975). Ionizing radiation singly not produces much chl mutation as combination treatment produces. Among the chemical mutagens, EMS is now being widely accepted as the most efficient and influential mutagen which induces highfrequency and wide spectrum of mutation. When EMS combined with radiation it not only causes synergistic effect but affect mutation in a specific ways. Singh (et al. 1999) reported that combined treatments of gamma rays and EMSwere most effective in producing chl mutation frequency than their individ- ual treatments in Vigna Chl mutationsinduced by EMS, gamma rays and other mutagens applied individually or in combination were reported by a Kumar (et al. 2009) and Gandhi (et al. 2014) in Vigna radiate, Bolbhat and Dhumal (2009) and Kulkarni and Mogle (2013) in Mac- rotyloma uniflorum (Lam.)  Verdc., Sharma et al. (2010) in Pisum sativum L. Gaur et al. (2013) in Capsicum ann- uum. Lower doses of gamma and gamma+EMS mutation frequency increases but it significantly decreased at the higher doses. Sharma (1970) reported that chl mutation frequency decreased at higher doses when calculated on segregating M1 familiesbasis. For both the treat- ment higher frequency of chl mutation with moderate doses of mutagens was observed. It seems that the strong mutagens reach their saturation point even at lower or moderate doses in the highly mutable genotype. With increase in dose further than a limit, the strong muta- gens become more toxic than the higher doses of rela- tively weaker mutagens and do not increase mutation frequency (Kolar et al. 2011). Moreover mutation fre- quency observed maximum in gamma+EMS treatment both in M1 and M2 generation in comparison to gamma. It may be due to EMS, a chemical mutagen which causes formation of new sites for mutation. So use of gamma followed by EMS suggests that the chemical mutagen is more efficient in inducing mutations of genes needed for chl development (Shah et al. 2006). In both the treatment, highest frequency of xantha mutant was observed, The highest frequency of xan- tha mutants ismay be due to the genes for xanthophylls development that are readily accessible for mutagenic action ( Similar reports were already given by Lal (et al. 2009), Khan (et al. 2005), Haq (1990) . These mutants could not survive more due to block in chl synthesis (Blixt 1961). In gamma, after xantha viridis was the sec- ond highest mutant, these mutant survived tillmaturity. Viridis attributed may be due involvement of polygene genes for the formation of chl. In combinedtreatment second highest mutant recorded was albino, these type of mutant formed may be due to deficiency ordegrada- tion of chl formation enzyme. in chickpea all chl mutants including albina type were in general morefrequent in EMS treatments than in gamma rays (Singh 1988). Chl is a vital biomolecule which plays a critical role in the life processes of allplants. Plants photosynthe- sis by absorbing light and transferring light energy to the reaction centers (chl molecule)of the photosynthetic system. Thus, Chl is essential for plant development and agricultural production (Eckhardt et al. 2004; Flood et al. 2011). Chl development seems to be controlled by many genes that are located on different chromosomal sites (Wang et al. 2013). It derivesby the formation of a long chain of biochemical process in which lots of loci were involved. The phenotypes of leaf color mutations are varied and are affected by different genetic and environment factors. Mutant plants leaf shows lower or higher chl content than normal leaf. This revealed that rate of change in the content of Chl a and Chl b was not the same among the mutants, possibly was due to the impair of Chl b synthesis during chloroplast develop- ment (Kolar et al. 2011). Ionizing radiation and chemical mutagen at higher concentration affectschloroplast thy- lakoid membrane which causes disability in chl manu- facturing. This chl deficiency reduced the rateof plant growth. Mutant plants with a higher (Nielsen et al. 1979) like viridis or a lower like chlorine, xantha (Vaughn et al. 1978; Wu et al. 2007) Chl a/b ratiothan that of their 24 Girjesh Kumar, Rajani Singh respective normal plants have been reported to be able to survive photoautotrophically. Mutations affecting the production of chl are important for identifying gene function and the elucidation of chl metabolism and its regulation (Wu et al. 2007). Cy tologica l investigation def ines t he specif ic responses of different genotypes to a specific muta- gen and it is also provides significant evidences for the selection of desirable traits (Kirchhoff et al. 1989).In the present appraisal chl mutant depicts various anomalies such as , stickiness, multivalent, precocious movement of chromosome, laggard and bridges. Stickiness could arise due to depolvmerization of ’ nucleic acid caused by mutagenic treatment (Avijeet et al. 2011). The forma- tion of multivalent (Fig. 6 G) may also be attributed to the abnormal pairing and non-disjunction of bivalents (Jabee et al. 2008). Jafri (et al.2011) suggested that pre- cocious movement (Fig. 6 D&I) of chromosomes was probably caused by spindle dysfunction. Laggard for- mation (Fig. 6F,K &L) is due to delayed terminalisa- tion, chromosomal stickiness or failure of chromosomal movement (Reddy and Munirajappa 2012). Due to direct action of mutagen target proteins gets defective and cre- ates the disturbance during chromosome separation and it forms bridges (Kumar and Gupta 2009). The incre- ment in the chromosomal aberrations might perhaps be due to the interactions of ionizing particles with the protoplasm, mediated through the excitation introduced by radiation that ultimately has increased the aberration frequency (Shukla nee Tripathi and Kumar 2010). When mutagens affects plant tissues internally it get accommodated and damage the cell or genes which- phenotypically can be seen. Genetically, during the M1 generation the probability of the occurrence of phe- notypicmutation is extremely low and only dominant mutations can be identified (Roychowdhury and Tah 2013). During the M2 generation, the chancefor identi- fying visible changes or phenotypic mutations should be higher and mostly due to genetics. Thereforeob- served mutations in the M2 generation are considered more stable (Parry et al. 2009). The plant height at low- er doses of gamma and gamma+EMS was significantly increased but higher doses of both the treatment causes inhibition /depression in plant growth. As according to Van Harten (1998) said, at high amount irradiation could cause the physiological damages such as inhibit cell division, death of cell and growth rate and genetic changes on the plants by producing free electrons radi- cal. Internodal length, leaf area and primary number of branches were increased at 100Gy dose of gamma. These characters were significantly higher at lower concentra- tion but at higher concentration it shows a reduction pattern. Treatment of gamma rays and EMS exhibited increase in mean values of number of primary branch- es in Lathyrus sativus (Waghmare and Mehra (2000). In mutation breeding programme, yield and its attrib- uted traits are very important parameters because ulti- mately breeders want to improve yield and related char- acters (Shahwar et al. 2020). Similar result was givenby Hanafiah (et al. 2010) in irradiated Glycine max (L.) Merr., var. Argomulto seeds with gamma rays shows- phenotypic variations that occur on M1 plants which affects plant growth development and production and also was given by Sinuraya (et al.2017) in Allium cepa assay. Thilagavathi and Mullainathan (2011)concluded that the decrease in quantitative traits have been attrib- uted to the physiological disturbance or chromosomal damage caused to the cellsof the plant by the mutagen as Williams (et al.1990) observed that due to nucleotide substitutions and insertion or deletions polymorphism occurred between individuals. In comparison to con- trol treated plant at lower dose (100Gy,200Gy, 100+0.1%) flowers early and matures more rapidly. At 200Gy+0.1% dwarf mutant with early maturing plant was identified. Panigrahi (et al.2015) suggested that significant varia- tions in quantitative parameters may showstable gene mutations in the next generations. Konzak (et al.1969) in wheat and Shakoor (et al. 1978) in Triticale reported that polygenes are responsible for semi dwarf character. Qin (et al.2008) reported dwarf rice mutants caused by single gene. Increased height and number of branches were due to loss of apical dominance which leads to lateral trans- port of growth hormone which results increased number of branches and bushy appearance as also observed ear- lier in Vicia faba (Shahwar et al. 2017), lentil (Solanki et al. 2004). Physical mutagen causes random change in the growth regulatory genes of plants but chemical mutagen exactly targets their mutagenic site through point muta- tion. This is the reason combination treatment proved to be more mutagenic and produces good amount of mutants. The selection of effective and efficient muta- gens is most important to recover the spectrum while high frequency of desirable mutations and efficiency of a mutagen indicates relatively less biological damage in relation to induced mutation (Solanki and Sharma 1994). CONCLUSION This investigation revealed the potency of gamma and in combination with EMS ,on increasing genetic diversity and demonstrated the successful program of induced mutagenesis in the Artemisia annuaL. In this breeding programme a total of 44 mutant in gamma and 25Analyzing frequency and spectrum of chlorophyll mutation induced through Gamma ray and Combination treatment 67 mutants in combination treatment were segregated. Various chl variants were identified in treated sets. Xan- tha was predominant among all the variants as most of the leaves found pale yellow colour. It is noticed that these changes are differentially sensitive to gamma and gamma+EMS and the appearance of new mutants would very helpful in maintaining the genetic purity of plant variety. So it should be important to identify desirable mutant plants through isolation and selection method. The cytological analysis of these mutants showed that these changes were induced due to changes in chromo- some number, structure, base substitution and dele- tion. For Artemisia LD50 recorded as 200Gy. 100Gy and 100+0.1%EMS was noticed good for plants growth and development as plant height, internodal length, leaf area and primary number of branches improved. So gamma and gamma+EMS induced reasonable chl mutations, hence all these treatments could be used in mutation breeding programs for inducing viable mutations only threshold dose of mutagen should be identified. 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