New Plant Type Mutants in Jute (Corchorus olitorius L.)

Three macromutants namely, ‘pigmented stem’, ‘viridis’ and ‘lax branching’, of jute (Corchorus olitorius L. JRO 524; ­commercial­fibre­yielding­crop)­were­induced­at­M2­following­gamma­irradiations­and­EMS­(Ethyl­Methane­Sulphonate)­ treatments.­The­true­breeding­mutants­(selfed­seeds­of­a­single­phenotypically­stable­plant­ forwarded­ in­each­generation­from­M4)­are­assessed­considering­different­attributes­like­germination,­survivability,­meiotic­chromosome­behavior,­ pollen­fertility­and­viability­and­quantitative­traits­across­the­generations­(M5,­M6­and­M7)­in­relation­to­control­under­a­uniform­field­condition(s).­Further,­genetic­variations­between/among­the­mutants­and­between­mutant(s)­and­control­is­also­ analyzed­from­seed­protein­(SDS-PAGE­-­sodium­dodecyl­sulphate-polyacrylamide­gel­electrophoresis),­RAPD­(Random­ Amplification­of­Polymorphic­DNA)­and­ISSR­(inter-simple­sequence­repeat)­profiles.­Pigment­profiles­following­TLC­(Thin­ Layer­Chromatography)­have­been­studied­in­‘viridis’­and­control.­Inheritance­of­the­mutant­trait(s)­has­also­been­assessed­ from­reciprocal­crossings­performed­between­control­and­mutants.­Results­suggest­that­the­mutants­are­true­breeding,­ stable­and­significant­and­may­enrich­genetic­resources­in­the­species.


Introduction
Induced mutagenesis is a potent tool for widening gene pool in a species through creation of genetic variations in a short span of time. Experimentally induced mutation has contributed significantly to global agriculture by producing more than 3000 mutants with desirable qualitative and quantitative traits in about 175 crop species 1 . Jute is an important crop species (Corchorus capsularis L -white jute, C. olitorius L. -tossa jute; once classified with the family Tiliaceae, more recently with Malvaceae, and has now been reclassified as belonging to the family Sparrmanniaceae) yield fiber of commerce (common name pat 2 ) from bark of the stem (phloem fiber). A good number of elite 'plant type' mutants are reported in C. capsularis [3][4][5][6] as well as in C. olitorius 4,5,7 but it is always desirable to raise superior mutants of economic worth by exploring the existing germplasm(s).
Maity and Datta 8 raised 3 plant type mutants in C. olitorius namely, 'pigmented stem' , 'viridis' and 'lax branching' following gamma irradiation and EMS treatments. Present communication describes the mutants in advance generations (M 5 to M 7 ) upon considering parameters namely, germinability, morphometric traits, meiosis, pollen fertility and viability and seed viability for ascertaining stability of the raised plant types. Further, seed protein (SDS-PAGE -sodium dodecyl sulphatepolyacrylamide gel electrophoresis) profile and RAPD and ISSR (Inter-Simple Sequence Repeat) molecular markers are used for genetic characterization of the mutants in relation to control. The objective of the work is to recommend new, stable and true breeding germplasm resources in C. olitorius that may be beneficial for commerce as well as for efficient breeding.
Viability of the pollen grains in each plant type was tested using stain tests namely, aniline blue in lacto phenol (detects the presence of callose on pollen wall, viable pollen turns blue 11 ) and FDA -Fluorescein Diacetate (detects presence of cytoplasmic starch that cleaves moieties from lipid soluble non-fluorescent probe to yield fluorescent product 12 ). FDA analysis was made under fluorescence microscope (Olympus, model: MLXi with Micro-LED fluorescence attachment; excitation maximum 475 nm, emission maximum 535 nm).
Photomicrographs in all cases were taken and subsequently magnified.

Crossing Experiments
At anthesis (9 am to 11 am) reciprocal crossings between control and mutants (5 crosses in each set) were performed following all necessary precautionary measures to avoid genetic contaminations. Hybrid seeds obtained from crossings were sown to raise F 1 plant population and subsequently F 2 (5 floral buds at F 1 were bagged in each category) plants. The F 2 plants segregated into normal and mutant phenotypes and χ 2 test analysis has been performed to assess segregation patterns.

Seed Protein Analysis
Quantitative analysis of seed protein of the plant types was made following the extraction method suggested by Osborn 13 and estimation by Lowry et al. 14 .

SDS-PAGE
The seed protein of 3 mutants along with control was used to study one dimentional SDS-PAGE (10% separating gel; 4.5% stacking gel) following Laemmli 15 in a vertical gel system (BIOTECH, Yercaud-Salem). For the purpose, total seed protein was extracted in 0.2 M Tris buffer (pH 8.5), suspended overnight (-4°C) and centrifuged at 15,000 rpm (-4°C) for 40 minutes. The protein samples along with sample buffer containing bromophenol blue were denatured in boiling water (1 minute), cooled and loaded in lanes with micropipette (12 µl/ lane; 0.075 µg/µl). A protein molecular weight marker (GENI Bangalore, Cat No. PMW-M) was also incorporated into the gel (as marker lane) as reference to detect molecular weights of the bands. The gel was run at 33 mA (3 mA/lane) for about 3 hours, stained in Coomassie contamination) along with selfed control lines (C. olitorius L. -JRO 524; mother seed stock obtained from CRIJAF -Central Research Institute for Jute and Allied Fibres, Nilganj, Kolkata, West Bengal, India in the year 2006) were used as germplasm source. In each generation, the plant types were grown in randomized block design with 3 replications (plot size: 3 m × 1.5 m, between plants 30 cm and rows 20 cm) in the experimental field plots of Department of Botany, University of Kalyani (West Bengal plains, Nadia; latitude 22°50´ to 24°11´ N, longitude 88°09´ to 88°48´ E, elevation 48 feet above sea level, sandy loamy soil, organic carbon 0.76%, soil pH 6.85) as rabi crop from March to October (2010-2012). No fertilizer application was made during growth periods of the plant types.

Quantitative Parameters
Germination frequency, quantitative morphological parameters and seed viability were analyzed in the plant types across generations. Morphometric quantitative traits were assessed on harvest. On an average 20 to 25 randomly selected plants were analyzed for quantitative parameters in each plant type and in each generation. χ 2 test of heterogeneity (DF 2) has been performed for each quantitative trait to assess variations, if any, among the years. Viability of seeds was tested following 1% tetrazolium chloride method 9 .
Fibre yield in the plant types was estimated following the conventional method of whole plant retting. For this purpose, defoliated plants of mutants and control were dipped in stagnant pond water. Brick bats tied in cement bags were used to deep the jak materials. Retting was completed within 20 days and fibre has been extracted by "beat-break-jerk" method.

Meiosis and Pollen Grains Attributes
For the meiotic studies, 3 to 4 floral buds of each plant types (control and 3 mutants) of each generation (M 5 to M 7 ) were fixed (6 am to 7 am) in Carnoy's solution (6 ethanol : 3 chloroform : 1 acetic acid) and 2 to 3 changes were given in the fixative at an interval of 24 hours and preserved in 70% alcohol. Pollen mother cells (PMCs) and pollen grains obtained from anther squash preparations were stained in 2% propinocarmine solution. Fully stained pollen grains were considered fertile 10 . Data were scored from scattered diplotene, metaphase I (MI) and anaphase I (AI) cells and pooled over the buds for each plant type in each generation. band(s) of individual primer, average polymorphic band per primer and polymorphism percentage. Shannon's diversity index 18 was calculated for each primer across the plant types.

Chlorophyll Estimation and Pigment Profile by TLC
Chlorophyll was extracted and estimated from leaf (1 gm for each sample, 3 replicas) tissues (of identical maturity) of control and 'viridis' (M 7 generation plants) following the method Arnon 19 . The extraction of chlorophyll and carotenoid pigments for Thin Layer Chromatography (TLC) was performed as per Pavia et al. 20 . TLC was done from the extracted samples in triplicate as suggested by Fried and Sherma 21 . The mobile phase used in the chromatography was pet. ether : isopropanyl alcohol : water (9:1:5 drops). After complete run, the TLC plates were taken out of the chambers, air dried and spots of different colorations were observed and photographed. Rf values were calculated and compared with reference chart provided by Pavia et al. 20 for quantitative plant pigment analysis.

Analysis of Quantitative Parameters
Data scored for morphometric traits of the plant types across the generations are presented in Table 1. Results indicated that 'lax branching' mutant ( Figure 1d) is a productive plant type possessing enhances number of branches, seed and fiber yield per plant. 'Pigmented stem' (Figure 1b) is also with higher number of seeds/capsule and enhanced seed yield/plant. Apart from the significant marker color trait in 'viridis' plant type (Figure 1c), quantitative parameters are either less or comparable to control ( Figure 1a). Analysis of each parameter in each Brilliant Blue R250 for overnight, destained and stored in 7% acetic acid 16 . Gels were scored and analyzed in gel documentation unit (Ultra Lum, USA) using the software TotalLab TM Quant v 11.

Molecular Analysis
Total genomic DNA from 4 plant types (3 mutants and control; harvested seeds of a single plant of the mutants were used in each case in M 7 ; control-seeds from selfed lines of 2012) were isolated separately from 0.2 gm of germinated seedlings using DNeasy Plant Mini Kit of Qiagen, USA. The seedlings were raised in Petri plates lined with moist filter papers (27° ± 1°C). The mucilage and polyphenolic compounds were removed by passing DNA through an Au-Prep purification column (Life Technologies, Rockville, MD, USA). Subsequently, quality and quantity of extracted DNA were checked by running the dissolved DNA in 0.8% agarose gel by comparison with standard lambda DNA marker of known concentration. The DNA was diluted to 30 ng/μl for RAPD and ISSR analysis.  17 . Three repeat runs were made and the primers documenting uniformity in band position and intensity were considered for analyses.

RAPD and ISSR Analyses
RAPD and ISSR bands were designated based on their molecular weight and calculated using the kilobase (kb) ladder used as marker. Molecular data were processed in MS excel for calculating polymorphic plant type across generations suggests randomness (P>05, DF 2) in distribution. All mutants bred true across the generations.

Meiosis and Pollen Attributes
Meiotic configurations, pollen fertility and pollen viability of the plant types are presented in Table 2. The meiocytes had 2n=14 chromosomes always ( Figure  2a-d). Assessment of meiotic configurations at diplotene, MI and AI indicated that it is comparable among/ between plant types as well as among generations of each plant type. Bivalent configuration is predominantly rod rather than ring. Univalent frequency (0 to 14/cell) is found to be higher in 'pigmented stem' than other plant types (Figure 2c

Seed Protein Profile by SDS-PAGE
Seed protein profile following SDS-PAGE (Figure 3) of C. olitorius (control) and 3 mutants (Table 3)

Chlorophyll Content and Pigment Profile in Control and 'Viridis'
Chlorophyll content (mg/gm) is found to vary significantly (

Discussion
Analysis of quantitative parameters in control and mutants across the years suggested that 'lax branching' mutant is a productive plant type as it possesses enhance number of capsule per plant and seed and fibre yield per plant compared to control. Capsule per plant and seed yield per plant are also found to increase in 'pigmented stem' . Estimation of chlorophyll content and preliminary analysis of pigment profile following TLC revealed significant variations between 'viridis' and control plant types. Leaf and stem colors of the raised and maintained 'viridis' and 'pigmented stem' mutants respectively are important as they may be explore as genetic markers in efficient breeding program of C. olitorius. Random distribution of quantitative traits, high frequency of 7II formation in meiocytes along with high pollen fertility and viability in mutants across generations suggested stability of the plant types. Further, seed germination frequency and seed viability percentage in mutants indicated that they are well acclimatized under the grown environmental condition(s). Macromutants namely, 'crumple leaf ' and 'tobacco leaf ' 7 , 'narrow leaf ' 4 , 'KOM 62' 5 among others have been reported in C. olitorius. Thus, the mutants described are new to the species. Lax branching and pigmented stem traits segregated at F 2 to a close fit of 3:1 ratio thereby, indicating monogenic recessive inheritance of the mutant traits. However, 'viridis' showed digenic (15:1) mode of inheritance. Results of reciprocal crossings are found to be similar, which suggested that the trait(s) are under nuclear gene control. Most mutation involving qualitative traits in C. olitorius are reported to be monogenic recessive excepting for 'leaf surface' , 'rolled leaf ' and 'fuzzy seed coat' , which showed digenic mode of inheritance 22 . Hazra and Karmakar 22 also reported that genetics of anthocyanin pigmentation in the species is rather complex and seems to involve 56 genes.
Apart from possessing lax branching nature and seedling and stem color traits, the mutants are concomitantly associated with other qualitative and quantitative traits. Kreft et al. 23 suggested that high lysine genes in barley and genes for the determinant growth in buck wheat (Fagopyrum esculentum) were suitable examples to show that the effect of single mutation could be complex or 'pleiotropic' . Pleiotropic gene action was found to influence stem, petiole, and bract tip pigmentation of sunflower 24 . Stearns 25 suggested the potential significance of pleiotropic gene action on evolutionary biology.
Seed protein content does not vary significantly among the plant types. However, seed protein profile following SDS-PAGE showed mark variations among/between plant types. Specific polypeptide bands are identified in control as well as in different mutants, apart from possessing common bands. Seed protein expression resulting in the formation of polypeptide bands is considered to be dominant over non-expression 26 . Mutation in regulatory and/ or structural genes may lead to failure of protein expression 27 . In the context, it may be inferred that presence or absence of polypeptide band(s) in different mutants in relation to control can be use as reliable marker for their identification and preparation of protein profiles.
Molecular analyses of the plant types using RAPD and ISSR markers indicated distinctive genetic variations (variation in the number of fragments generated)  between/among the mutants as well as between mutants and control. Average polymorphism percentage is found to be 46.45% by RAPD and 57.14% by ISSR primers. RAPD markers generated wide range of variations (11.11% to 92.31%) than ISSR (42.86% to 72.73%) across the plant types. RAPD markers are used to assess genetic variations caused due to mutation in different plant species [28][29][30] .
Sen and Alikamanoglu 31 performed analysis of drought tolerant sugar beet (Beta vulgaris) mutants induced with gamma irradiation using SDS-PAGE and ISSR markers and found wide range of polymorphism in the band profiles.

Conclusion
The macromutants are true breeding and stable. Seed protein analysis by SDS-PAGE and molecular analyses following the use of RAPD and ISSR markers suggests distinctive genetic variations between/among the plant types. 'Lax branching' mutant is found to be a productive in relation to seed and fibre yield, although plantation per unit area may not be promising. 'Viridis' and 'pigmented stem' mutants possessing genetic marker trait(s) can be important resources in efficient breeding program in C. olitorius. The mutants may be recommended as 'new plant types' , enriching genetic resources in the species.