VEGETOS: An International Journal of Plant ResearchOnline ISSN: 2229-4473
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Research Article, Vegetos Vol: 30 Issue: 3

Development of Early Maturing Linseed Genotypes Through Induced Mutatagenesis Suited for Cultivation in Rice Fallows

Suma Mogali*, Lalita Jaggal, Yamanura and Revanappa Biradar

AICRP on MULLaRP, University of Agricultural Sciences, Dharwad, Karnataka India

*Corresponding Author : Suma Mogali
AICRP on MULLaRP, University of Agricultural Sciences, Dharwad, Karnataka India
Tel: 91-836-2214420
E-mail: [email protected]

Received: March 31, 2017 Accepted: May 18, 2017 Published: May 22, 2017

Citation: Mogali S, Jaggal L, Yamanura, Biradar R (2017) Development of Early Maturing Linseed Genotypes Through Induced Mutatagenesis Suited for Cultivation in Rice Fallows. Vegetos 30:3. doi: 10.5958/2229-4473.2017.00150.1

Abstract

A substantial part of rice fallows during the rabi (post rainy) season are left uncropped because of limited availability of soil moisture. Late maturity is a major inconvenience to utilize the residual moisture after paddy harvest, where plants must mature within a much shorter growing season. The objective of this study was to develop early maturing linseed mutants within the 100 days for efficient utilization of available residual moisture after paddy harvest. The experimental material was developed by involving a promising variety NL-115 which was treated with 0.1,0.2, 0.3 and 0.4 per cent EMS for 18 hours. Subsequently from the M2 generation, plants were selected for early maturity, compact growth habit coupled with higher seed yield. From the wide array of mutants generated in this study new mutants exhibiting early seed maturity coupled with high seed yield, lodging tolerance were isolated. Successful development of useful mutants in linseed was reported before, but this is the first report on successful use of mutation breeding for improvement in quantitative traits in this crop..

Keywords: Linseed; Early maturity; Induced mutagenesis

Introduction

Rice-fallows are lands used to grow rice in the kharif season but left uncropped during the following rabi season. A substantial part of this area remains fallow during the rabi (post rainy) season because of several limitations, the prime one being limited availability of soil moisture. More specifically the constraints and opportunities for farmers in similar situations are expected to be identified to make better use of their land by growing short-duration crops with minimal inputs in the rabi season on residual moisture after kharif rice has been harvested. For this reason introduction of rabi crops on residual moisture can be expected to bring a substantial improvement of farmers economic conditions in the marginal lands of these poverty ridden and deprived regions. Among the oilseed crops grown during rabi, linseed is next important to Rape seed mustard in area as well as in production. Linseed is grown in area of 2797 thousand hectares with an annual production of 16, 02,047 metric tonnes of seeds [1]. Canada is the major linseed producing country along with Argentina, India, USA and Russia. India is the second largest producer of linseed, with an area of 296.3 thousand hectares, a production of 148.6 thousand metric tonnes per annum and productivity of 502 kg/ha. India has 18.8 percent of world’s recorded linseed area but produces less than 10 percent total world production. Efforts need to be directed towards yield enhancement.

Flaxseed is the richest source of plant lignans Secoisolariciresinol diglucoside (SDG), which are phytoestrogens and serve as precursors in the production of mammalian lignans. It has the antioxidant activity and thus may contribute to the anticancer property of flaxseed, lignans could influence bone development and also have been shown to be protective against breast cancer [2].

Regardless of its multiple and varied uses linseed is one of the neglected oilseed crops of developing countries, grown on marginal land with poor management. A poor yield of this crop is attributed to non-availability of improved cultivars to suit the diverse agro climatic condition. Hence, development of high yielding cultivars with improved oil quality becomes the top priority to overcome the poor yield levels and quality.

Assessment of genetic variability is the first step in any crop improvement programme. It may be created through hybridization or induced mutation. Since linseed is a self-pollinated crop and a strong negative association between some of the yield components due to linkage and pleiotropy [3] is a bottle-neck in realizing the predicted genetic advance under selection expected on the basis of performance of early generations in a hybridization programme. Therefore, induction of mutation and their utilization through effective selection sieves in the altered linkage situations appeared to be a reasonable approach in improving the yield potential of this crop [4]. Further rice-fallows are lands used to grow rice in the kharif season but left uncropped during the following rabi season. A substantial part of this area remains fallow during the rabi (post rainy) season because of several limitations, the prime one being limited availability of soil moisture. More specifically the constraints and opportunities for farmers in similar situations are expected to be identified to make better use of their land by growing short-duration crops with minimal inputs in the rabi season on residual moisture after kharif rice has been harvested. For this reason introduction of short duration rabi crops on residual moisture can be expected to bring a substantial improvement of farmers economic conditions in these poverty ridden and deprived regions. Another problem of growing linseed in northern transitional tract of Karnataka, India is lodging problem owing to the higher plant height and lower stem girth. Therefore the current study is conceived with the main focus to develop linseed mutants, which possess early maturity, compact growth habit and higher seed yield, suited to be grown in paddy-fallow eco-system.

Methodology

The present experiment was conducted during three seasons viz., rabi, 2012-13, 2013-14 and 2014-15 at Main Agricultural Research Station (MARS), University of Agricultural Sciences, Dharwad, which is located at latitude 15°26' N, longitude of 75°07' and situated at an altitude of 678 m above mean sea level, which falls under agroclimatic zone-8 (North Transitional Zone) of Karnataka.

The material for present study was generated at Main Agricultural Research Station, University of Agricultural Sciences, Dharwad. The experimental material was developed by involving a linseed variety NL- 115. Although NL-115 variety of linseed is a good yielder, this cultivar has duration of 110 days with a plant height of 45-55 cm and erect growth habit and produces yield of 840 kg/ha and 1000 seed weight of 6.2 g. The maturity duration of 110days is a problem as the crop suffers from terminal drought when sown on residual soil moisture. By contrast, short duration varieties which can efficiently utilize the available soil moisture are best suited for paddy fallow ecosystem.

The pre hydrated seeds (8 hours) were treated with chemical mutagen, ethyl methane sulfonate (EMS) solution which was prepared in 0.1 M phosphate buffer (pH-7.0), at five different doses ranging from 0.1 to 0.5 percent. Later the seeds were washed in running water to remove chemical residue and shade dried before sowing.

Raising M1 and subsequent generations

The treated seeds were sown in the field with an inter-row spacing of 30 cm and intra row spacing of 10 cm during rabi 2012-2013 along with respective control to raise M1 generation. All the recommended agronomic and plant protection practices were followed to raise the crop. The surviving plants were harvested separately to raise M2 generation (Figure 1).

Figure 1: Variation for Flowering time and maturity between the control and the mutant.

The seeds harvested from individual progeny row in M1 generation were utilized to raise M2 generation. However, no progenies were advanced from 0.5 per cent EMS treatment as no desirable progenies were observed. Similarly, their untreated counterparts were sown in adjacent lines for calculation of environmental variance. Spacing of 30 × 10 cm was followed between inter and intra rows. The standard agronomic practices were followed to raise a good crop. At the time of harvest, observations were recorded on around 20 randomly selected plants each from treated and untreated parents for yield and yield related characters. In M2 generation, desirable mutants were selected from 246 progenies consisting of 4712 plants. Breeding behaviour of the mutants was confirmed in M3 and M4 by growing progeny to row method.

Morphometric observation

Except for days to 50 per cent flowering, all other observations were taken at maturity. The mean value for individual mutant line was computed by taking average in M3 generation. Observations on characters such as days to 50 per cent flowering, days to maturity, plant height at maturity (cm), number of primary branches per plant ,number of secondary branches per plant, number of capsules per plant, number of seeds per capsule, test weight oil content and fatty acid composition were recorded.

Oil content and fatty acid composition of seeds was measured using Near Infrared Spectroscopy (NIRS) at Seed Quality and Research Laboratory, UAS, Dharwad. The instrument was initially calibrated and validated using the fatty acid profile data of linseed core collection provided by Dr. N.Y. Kadoo of National Chemical Laboratory, Pune (Figure 2).

Figure 2: Erect and non-lodging mutant.

For NIRS analysis, samples of well matured seeds were taken from each plant and used for fatty acid analysis. About 2g of seeds were placed in a special adapter about 3 mm thick, with a diameter of 15 mm. Before spectra acquisition, a reference spectrum was collected from a standard check cell (IH-0324A, Infrasoft International, LLC, France). The instrument diagnostics was carried out to test the response of instrument, wave length and NIR repeatability to avoid the effect of surrounding environment on the instrument performance. The absorbance spectra (log 1/R) from 400 to 2500 nm were recorded at 8 nm intervals. Mathematical procedures on the spectral information were carried out with WinISI II Project Manager Software, Version 1.50 (Infrasoft International, LLC).

Statistical Analysis

The data recorded were processed with the help of Ms-excel and Windostat version 8.1 available at Department of Genetics and Plant Breeding, University of Agricultural Sciences, Dharwad. The statistical parameters viz., range, mean, variance, standard error, coefficient of variation and other variability parameters were computed for all the traits in M2 generation and M3 generation (Table 1).

  M1 M2 M3
Treatment Number of Seeds treated Number of Plants harvested No of plants raised Number of variants observed Mutation frequency True breeding mutants
Control 1000 905 - - - -
0.1% EMS 1000 655 906 9 0.99 17
0.2% EMS 1000 585 1228 19 1.54 16
0.3% EMS 1000 540 1188 18 1.51 11
0.4% EMS 1000 412 1395 36 2.58 7
Total       4717 82 1.7 51

Table 1: Effect of different concentration of EMS on linseed.

Results and Discussion

A total of 82 desirable variants were isolated from M2 population consisting of 19,619 plants. Out of 12 different treatments, treatment with 0.4 percent EMS was ideal with the highest mutation frequency of 2.58 percent in the M2. Usually EMS acts as a base substitution agent. During the metabolically active stage, presence of EMS hinders repairs in errors during replication during germination and mis- incorporation of bases occur at a high frequency leading to accumulation of mutation.

Among 82 variants in the M2, 51 true breeding mutants for plant height, plant habit, leaf related traits, flower kenel size and testa colour were identified in M3. Variability among the mutants was also observed in terms of days to maturity, number of primary branches, secondary branches, capsule number and size and thousand seed weight resulting in their enhanced yielding ability.

Short duration mutants

In the present study, promising mutants for early maturity (Mutant No. 66,) and ideal plant type (Mutant No. 78) with nonlodging ideotype (Figure 3). The mutant line 66 derived from 0.3 per cent EMS treatment recorded an early days to 50 per cent flowering and maturity at 35 days and 95 days respectively, compared to mean value of 45.20 days for 50 per cent flowering and 104 days for maturity in corresponding control (Table 2). This mutant has recorded significantly higher number of secondary branches per plant 75 compared to the parent with 23 secondary branches, significantly higher number of capsules per plant(230),while its non-mutant parent has recorded 56 capsules contributing to significantly higher yield of 15 q/ha compared to 7.35q/ha recorde by its parent. Other short duration mutants isolated from this study have been presented in Table 2. These genotypes with shorter duration are suited to be grown in paddy fallow ecosystem.

Figure 3: Variation of Capsule size.

Mutants Seed yield (kg/ha) Days to 50% flowering Days to maturity Plant height(cm) Number of primary branches/plant Number of secondary branches/plant Number of capsules/plant Number of seeds/capsule Test weight (g) Oil (%)
66-3 1500* 35* 95* 40 5 75* 230* 6 13.83 40.30
79-4 740 35* 98* 41 9* 73* 215* 5 11.83 40.43
40  
                   
60-4 655 35* 98* 38 12* 103* 438* 5 15.37 39.40
26-3 800* 35* 94* 36 19* 209* 325* 4 8.64 40.13
Parent Mean 735 45.2 110 46.64 4.37 23.25 56.01 6 9.45 39.74
CD at 5% 12.23 1.78 1.895 7.989 4.121 17.871 22.34 2.027 8.21 1.94

Table 2: Performance of early mutants for seed yield and test weight in m5 generation.

In linseed, due to lower stem girth, lodging is a common problem. The mutant 78-2 derived from 0.3 per cent EMS treatment exhibited an ideal plant type with erect growth habit and thicker stem as compared to corresponding control, thus avoiding the lodging of the plant. It is also characterized by increased number of primary branches (11), secondary branches (97), capsules per plant (203) and yield of 4.77 g. These mutants could be used in future breeding programmes aiming towards improvement with respect to these traits.

Large capsule mutants

It was interesting to observe that, a single capsule in a mutant derived from 0.5 per cent EMS treatment measured around 1.1cm whereas the rest of capsules in that parent measured 0.7cm. Capsule diameter increased by 57 percent in this mutant compared to the parent (Figure 4). Hence it was harvested separately and capsule to progeny row was raised in M3 generation.

Figure 4: Types of various mutants.

There were six plants raised from that single capsule and their performance for yield and yield attributes were recorded (Table 3). Variability was observed for capsule size, number of capsules, yield per plant and test weight in M3 progeny derived from large capsule mutant, this could be due to segregation in M3 generation for these traits for heterozygote loci. Breeding behavior of these variants are being ascertained in M4 generation during rabi-2015. Among the six plants, a mutant 106–2 recorded highest yield of 17.36 grams compared to all the mutants in the present study. Another two plants of the same progeny namely 106-4 and 106-3 recorded increased yield of 12.1 and 12.0 g respectively.

Mutants Days to 50% flowering Days to maturity Plant height Number of primary branches/plant Number of secondary branches/plant Number of capsules/plant Number of seeds/capsule Seed yield/plant (g) Test weight (g)
106-2 51 114 55 14 81 270 8 17.36 10.96
106-4 51 114 54 13 52 225 8 12.1 8.58
106-3 51 114 58 9 58 278 8 12.02 8.41
control Mean 45.4 104 48.04 5.2 27.11 56.07 6.6 3.51 10.46

Table 3: Performance of mutant number 106 (big capsule mutant) progenies in M3 generation for yield and yield components.

Superior mutants for seed yield

Yield increment being the main objective in most of the plant breeding programmes, mutation breeding has played a key role in achieving the goal. Few of the mutants in the present experiment produced higher seed yield compared to parents (Table 4). Among the superior mutants selected for seed yield, Mutant No.57-3 has recorded highest yield of 15.99 g per plant compared to 6.16 g in control. This considerable increase in yield is owing to increase in number of secondary branches (75), number of capsules per plant (230) and enhanced test weight of 10.25 g. Likewise in Mutant No.79- 4, which recorded 12.10 g, the increase in yield can be attributed to increase in number of secondary branches (73), number of capsules per plant (215) and mainly test weight (14.63 g) which is 1.54 times the control mean (9.45 g). Similarly in other high yielding mutants viz., Mutant No. 60-4 (10.66 g), 102-6 (10.25 g) and 53-3 (10.12g) the amplified yield can be owed to improvement in yield attributing traits like number of secondary branches, number of capsules per plant and test weight. Yield being a polygenic character, the yield advantage obtained might be due to the favorable gene mutation at more than one loci. Similar results were obtained by Badere and Choudary [5] where they isolated high yielding mutants with increased number of seeds per plant. According to them the increase in number of seeds was due to enhancement in the yield contributing characters such as, capsules per plant and seeds per capsule. Similarly Mohith chaudhary et al., [6] also reported that seed yield per plant (g) was recorded very strong positive association with number of capsules per plant, harvest index, number of secondary branches per plant, plant height and number of primary branches per plant. Path coefficient analysis revealed that harvest index followed by biological yield per plant and plant height emerged as most important indirect yield components (Figure 5). The improved mutants need to be evaluated for stability and if found consistently superior, they can be released as a variety upon large scale testing. Apart from the accessions identified which are suited to be grown in paddy fallow eco- system, the present study indicates that the mutagenesis using EMS has the potential to generate a large amount of variability and many useful mutants for increased test weight, seed yield per plant and other yield attributing traits like, number of primary branches, number of capsules per plant in M3 generation. Besides these variants, some morphological mutants were seen for petal colour and petal aestivation. Mutant number 26-1 produced white coloured petals and yellow kernels. (Parent possessed blue petals and brown coloured kernels). Such mutants with the changed kernel colour may also show distinct changes in fatty acid profile. Further such mutants will be tested for their consistent performance in the changed traits and analysed for their fatty acid profile EMS treatment causes alkylation of guanine bases (G) leading to mispairing or mismatch pairing in the DNA of a treated organism. Under these conditions, an alkylated G pairs with T (thymine) in place of C (cytosine), causing a G/C to A/T transition in the backbone of the DNA [7]. Selection of mutants through the subsequent generations may be helpful in eliminating deleterious mutations and recovery of genotypes with novel and desirable traits as we have observed that there is a recover of mutants with more number of capsules (298) in M4 generation as against the same mutant possessing 90 capsules in M3 generation, which contributes to increase in the yield per plant. A more detailed study is necessary to verify these observations in the subsequent generations. It is observed that, not a single genotype has recorded higher value for all these yield components. Identified mutants with important yield attributes can also be used as genetic stocks for linseed improvement. Two hundred and forty two mutants generated in the study which show wide range of variability for different qualitative and quantitative traits can be deposited after stabilizing them through two to three breeding cycles with International plant Genetic Resource Institute so that linseed researchers across the globe can utilize them to suit their local conditions,Further biochemical analysis including the proximate analysis of these mutants for composition of secoisolariciresinol diglucoside, motairesinol which have anti-oxidant potential, make linseed a source of wonder drug for ailments such as cancer. Flax seeds are also known for their prophylactic effect as they are rich source of ω-3 and ω -6 fatty acids. It is also known to lower cholestrol and blood pressure and prevent arthritis and cancer [8]. Therefore the mutants created in this study will be screened for high ALA through fatty acid profile analysis in M4 generation. It is quite possible that there may be changes in Alphalinolenic acid and lignan composition that could have resulted from pleotropic effects of mutation.

Figure 5: High yielding mutant and its parent. Besides these variants, some morphological mutants were seen for petal colour and petal aestivation.

Mutants Seed yield/plant (g) Days to 50% flowering Days to maturity Plant height Number of primary branches/plant Number of secondary branches/plant Number of capsules/plant Number of seeds/capsule Test weight (g) Oil(%)
57-3 15.99* 51 108 62 5 75* 230* 6 10.25 40.30
79-4 12.10* 51 108 53 9* 73* 215* 5 14.63 40.43
60-4 10.66* 49 105 52 12* 103* 438* 5 14.39 39.40
26-3 10.04* 51 105 59 19* 209* 325* 4 8.64 40.13
56-3 9.06* 51 110 67 9* 52* 256* 4 9.24 39.33
62-5 8.61* 51 106 59 16* 78* 287* 5 7.37 39.50
84-3 8.60* 48 108 51 23* 180* 674* 5 12.89 39.80
105-1a 8.58* 51 108 52 15* 120* 250* 6 6.89 39.80
Parent Mean 6.16 45.2 104 46.64 4.37 23.25 56.01 6 9.45 39.74
CD at 5% 1.223 1.78 1.895 7.989 4.121 17.871 22.34 2.027 8.21 1.94

Table 4: Performance of Superior mutants for seed yield and yield components in M3 generation.

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