SEED GROWTH AND SEED QUALITY IN DIPOGON LIGNOSUS (L.) VERDC. BEAN

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1 Legume Res., 6 (5) : 8-86, AGRICULTURAL RESEARCH COMMUNICATION CENTRE / indianjournals.com Received: -- SEED GROWTH AND SEED QUALITY IN DIPOGON LIGNOSUS (L.) VERDC. BEAN M.S.A. Fakir*, S.S. Das, and F. Islam Department of Crop Botany, Bangladesh Agricultural University Mymensingh-, Bangladesh Accepted:-6- ABSTRACT Lignosus (Dipogon lignosus) is valued for green seeds and young twigs as vegetable, dry split seeds as pulse soup and other plant parts as organic matter. Two field experiments were conducted and seed growth stages were investigated to determine physiological maturity (PM), time of harvest of green seeds for vegetable and to assess quality and protein content of seeds in Lignosus bean. Four consecutive stages of seed growth/maturity viz., prepm, PM, postpm and harvest maturity (HM) were considered. Effect of four seed growth stages on seed germination, seedling growth and protein content were investigated. Maximum seed fresh and dry weight (PM) was observed 4 days after flowering (DAF). PM to HM stages showed higher germination (> 8%) than PrePM (5.4%). Seed protein content was higher at postpm and HM (average of.6%) than in the others. It may be concluded that green seeds attained PM (4 DAF) with good seed protein content (.%) in Lignosus bean. Key words: Dipogon lignosus, Green seeds, Protein, Seed quality, Vegetable. INTRODUCTION Grain legumes are important sources of proteins and dietary fiber in addition to fats, carbohydrates and minerals. Lignosus bean (Dipogon lignosus (L.) Verdc.) is an important member of Fabaceae which is reported to be grown in the tropic and sub tropic as a minor field crop in Asia, Latin America, South America and Australia (Purseglove, 988; Fakir et al., 8). Due to extra hardiness and fibrousness of pericarp at maturity only the matured green seeds of Lignosus can be consumed as vegetables unlike Country bean (Lablab purpureus L.) where both pericarp and green seeds are soft. Lignosus bean is grown mainly for matured green seeds as vegetable, ripe seeds as pulse soup and vines as fodder (Fakir et al., 8; Fakir and Hasan, 9). vigour, germinability, green seed yield for vegetable, storability and different nutrient contents are at highest condition in the bean seed (Parashiva and Dushyantha, 4; Fakir and Abdullah, 7). Changes in seed mass, chemical composition and mineral concentration of developing seeds of common vetch (Vicia sativa L.) were studied by Nezar et al. (9) and showed that seed weight and protein content increased with aging and became highest at PM. Fakir and Abdullah (7) observed that at 4 days after flowering (DAF) all four indeterminate morphotypes of vegetable pigeonpea (Cajanus cajan) showed highest seed dry matter indicating PM. The authors further added that the germination ability, pod size and seed nutrient contents were also maximum at PM stage. In Lablab purpureus (cv. H.A.-) seed development and maturation was studied in India and PM was observed at 5 DAA (days after anthesis) with 97.% germination and 8.5% vigour index (Parashiva and Dushyantha, 4). According to Chinnasamy and Bal (), accumulation of weight (both fresh and Deposition rate of dry matter into the seed reaches maximum at a certain age after anthesis, known as physiologi cal maturity (PM). Determination of the right stage of PM is essential because at PM, dry matter content, viability and * Corresponding author s fakirmsa@gmail.com Bangladesh Water Developement Board, 49 Motijheel, Ansher Chamber, Dhaka Directorate of Secondary & Higher Education, Rajshahi Region, Rajshahi, Bangladesh

2 dry) in pod and seed, germination percentage, speed of germination, seedling length and weight, protein and lipid content in seed increased progressively with maturity and attained maximum value at PM stage in both beach pea (Lathyrus maritimus) and grass pea (Lathyrus sativus). In cowpea, appropriate time of harvest of vegetable pod and PM was observed at around days after flowering (DAF) and 5 DAF, respectively (Fakir and Hossain, ). Hasan (6) investigated seed growth and observed PM around 4 DAS in Lignosus bean. Introduction of Lignosus bean has been in progress in Bangladesh (Fakir and Hasan, 9). Although, information on some morphophysiological aspects, pod growth, and floral abscission, green seed storability is available (Fakir et al., 8; Rahman 8; and Fakir et al., 9), but there is scanty information on determination of PM stage, time of vegetable seed harvest, seed growth and quality of Lignosus bean (Hasan, 6; Das, 9,). Further, there is only one report on the effect of stage of pod/ seed maturity on germination and protein content of seed, and seedling growth in Bangladesh (Islam, 8). Therefore, the current research work was carried out i) to determine the stage of physiological maturity (PM); ii) to envisage the proper harvesting time of green seed for vegetable; and iii) to investigate the effect of stage of seed maturity on seed germination and vigour, and seed protein content in Lignosus bean. MATERIALS AND METHODS Two field experiments were conducted in the Field Laboratory and Botanical Garden, Department of Crop Botany, and protein was estimated in Nutrition Lab., Bangladesh Agricultural University, Mymensingh (4º75 N and 9 º 5 E) between 8 and 9. Plant culture and seed maturity (Expt. ): Two genotypes of Lignosus (n= ) bean viz., Trinidad and Chittagong were considered. The field experiment was laid out in randomized complete block design with three replications. Plants were established following the methods of Hasan (6) and Rashid (98). Seeds were sown in April, 8 and flowering began in January, 9. Opened flowers were tagged with coloured woolen threads at first opening of flowers, in order to collect data on pod and seed growth at, 5,, 9, 6,, 4, Vol. 6, No. 5, 8 47, and 54 days after flowering (DAF). For collecting data at each age (DAF), randomly harvested tagged pods were carefully brought to the laboratory and separated into samples ( pods/sample) with three replications. At each stage pod size (length and width) and weight (fresh and dry), and seed weight (fresh and dry) were recorded in the two genotypes. According to the visual index developed by Hasan (6), pods were harvested at four different and consecutive stages of seed growth/maturity for the genotype Trinidad only and was used in the Expt.. The first stage was prephysiological mature (prepm) seed stage with deep green colour pericarp and seed and the age was approximately -5 days after anthesis (DAA). The second stage was physiologically mature (PM) stage of around 4-45 DAA and with light green colour pericarp and seed. The third was yellowish stage after PM i.e. postpm, when pods were similar to PM but with yellowish pericarp and almost white seed. Harvest maturity (HM), completely dry seeded brown pod was the fourth stage. Germination and seed quality (Expt. ): The second field experiment was carried out from February to March, 9 in the Botanical Garden, Bangladesh Agricultural University, Mymensingh to investigate the effect of those stages of pod harvest or seed maturity on germination, vigour, seedling growth and protein content. Four stages of pod/seed harvest (prepm, PM, postpm, and HM) from the Expt. were collected and used in the Expt.. The pods were shelled and seeds from those four maturity stages were used as four treatments in the Expt. The experiment was laid out in a randomized complete block design (RCBD) with four replications. The recorded data were: initial seed moisture content, seed length and breadth, germination and vigour % at days after sowing (DAS); at DAS seedling weight per -seedling (fresh and dry), shoot and root length, seedling diameter (.5 cm below the cotyledon), leaf number per -seedlings (mature, fully expanded and immature, unfolded and expanding one recorded separately), leaflet number per -seedling (mature and immature), leaf area per -seedling, leaf weight per -seedling (fresh and dry) and leaf chlorophyll content. Leaf area was measured by leaf area meter (model L-, Licor, USA) and chlorophyll content was measured by

3 8 LEGUME RESEARCH Chlorophyll meter (SPAD-5, Konica Minolta Sensing, Inc. Japan). Percentage vigour was calculated as the sum of the percentage of seed that sprouted in a particular period of 4 hours, each divided by the number of day(s) after sowing. Thus, vigour rating for a batch germinating a, b, c k % on st, nd, rd.. and th days is a b c k.... Mature leaflet numbers (MLN) were counted as follows: MLN= + number of mature leaves; where the number represents first two simple leaves and are considered as two leaflets and each matured leaf consists of three leaflets. Immature leaflet numbers (ILN) were counted as: ILN = number of immature leaves. Total soluble solids (TSS) of fresh seeds were recorded by Refractometer (Model No.N-E, Series -W, Atago, Japan) at º C temperatures in % brix unit. The crude protein of seed at four maturity stages was determined (AOAC, 99). Statistical analysis: Different ages (days after flowering, DAF) of the pods were used as treatments. As such eight ages of seeds (DAF) were considered as treatments in the Expt.. In the Expt. four seed maturity stages (PrePM, PM, PostPM and HM) were used as treatments. In both the experiments a randomised complete block design was followed. Analyses of variance of different variables were performed. The mean differences were evaluated by least significance difference and Duncan Multiple Range Test (Gomez and Gomez, 984). RESULTS AND DISCUSSION Pod length and width: Generally Pod length increased slowly up to 5 days after flowering (DAF) followed by a rapid and linear increase up to 6 DAF (Fig. A). Between and 47 DAF pod length attained a plateau in the two genotypes. At 4 DAF the longest pod was observed in Chittagong (6.8 cm) genotype and shortest in Trinidad (6.45 cm). Pod width at different DAF followed a trend also similar to that of pod length (Fig. A). Pericarp and seed mass: Increase in fresh weight of pericarp was very slow up to 5 DAF which grew rapidly and linearly between and 6 DAF and reached maximum at DAF (.64 g in Trinidad and.6 g in Chittagong respectively), followed by a decline afterwards (Fig. B). The pattern of change in dry weight of pericarp was similar to that of fresh weight. There was little seed fresh weight up to 9 DAF which increased sharply and linearly between 6 and 4 DAF with a decline afterwards (Fig. C). At 4 DAF, the fresh weight of seed was greater in Chittagong genotype (.9 g/pod) than Trinidad genotype (.6 g/pod). The pattern of seed dry weight followed the trend of a sigmoid curve. Changes in dry weight of seed were similar to that of fresh weight (Fig. C). At 4 DAF maximum dry weight (. g in Chittagong and.97 g in Trinidad) of seed was found indicating physiological mature (PM) stage. Pod weight: Total fresh weight of pod (pericarp plus seed) increased slowly up to 5 DAF followed by linear and sequential increased between and 4 DAF and reached maximum to 5.4 g in Chittagong and 4.69 g in Trinidad genotype at 4 DAF with slight decline afterwards. Pod dry weight continued linearly up to the maximum (.78 g in Chittagong and.4 g in Trinidad) at around 4 DAF (Fig. D). The PM is usually used to denote maximum dry mass (DM) accumulation followed by high germination and vigour in the grain and seed. In present study, maximum DM accumulation was achieved around days after anthesis (DAF) in pericarp and that of seed at around 4 DAF in all the beans (Fig. ). This indicated that PM of Lignosus bean seed was at around 4 DAF in the two genotypes investigated. In Lignosus bean (Trinidad genotype), Hasan (6) studied seed development and maturation and showed that PM was obtained around 4 DAF. Thus, the current result on PM stage at 4 DAF in Lignosus bean was almost similar to that of Hasan (6). Based on increase in size and weight of seed and pod, the optimum picking time and, therefore, the most appropriate harvesting time is needed to establish in Lignosus bean for vegetable purpose. In the present investigation, in the two genotypes maximum length and width of pod were obtained at around 4 DAF and so was in case of weight. Maximum fresh weight was observed at DAF in pericarp but 4 DAF in seed. Fresh weight of pod also attained maximum at 4 DAF indicating the appropriate harvesting time of the green pod. These results further indicated that pericarp gained maximum fresh weight earlier than

4 Vol. 6, No. 5, 8 8 Le ngth (Trinida d) Width (Trinidad) Le ngth (Chittago ng) Width (Chittago ng) Fres h (Trinidad) Fres h (Chittago ng) Dry (Trinadad) Dry (Chittago ng) 7 A.5 B Pod length or width (cm Pericarp weight /pod(g F re s h (Trinida d) Dry (Trinada d) Fre s h (Chittago ng) Dry (Chittago ng) 6 5 D Seed weight/pod (g C Pod weight (g/pod Days after flowering (DAF) Days after flowering (DAF) FIG. : Changes in pod length and width (A); pericarp (B); seed (C) and pod (D) weights at different ages (days after flowering, DAF) in the two genotypes of Lignosus bean (Expt. ). Four vertical bars in each graph indicate LSD.5 values for four lines, respectively. seed. Moreover, the pericarp began to decrease weight after DAF indicating beginning of losses of green color along with seed shrinkage from pericarp, which was clearly observed at 4 DAF. Considering greenness and weight, appropriate time of harvest of Lignosus beans (both Trinidad and Chittagong genotypes) may be after DAF but on or before 4 DAF. The present result agrees with the reports of Hasan (6) that the right stage of harvestable vegetable seeds are fully developed bright green when the pods start loosing colour in Lignosus bean (Trinidad genotype). In a study with common vetch (Vicia sativa L.), Nezar et al. (9) also observed such fading of green color from pod at around PM.

5 84 LEGUME RESEARCH Seed maturity stages and seed quality: Initial seed moisture percentage progressively decreased with the increasing maturity (Table ). There was no significant difference in initial seed moisture content among the prepm, PM and postpm seed (average of 6.%) and this was significantly five fold higher than HM seed (.7%). The germination percentage differed significantly (P >.5) due to stage of seed maturity (Table ) and it was greater at HM (84.55%) than at PM (8.%) and prepm (5.4%). The germination percentage was greater than 8% and similar between HM and postpm (average of 8.7%). The vigour increased with increasing maturity and peaked at postpm (7.9%) followed by a decline in HM (45.%). In the present study, seed maturity category postpm (7.9%) had significantly higher vigour than PM (55.8%), HM (45.%) and prepm (.79%). Seed protein content was significantly greater at postpm and HM and (average of.6 %) than prepm (.5%) and PM (.%) (Table ). Total soluble solids (TSS) followed a trend almost similar to protein. Seedling fresh weight (per -seedling) was significantly higher at HM (9.8 g) than at PM (7. g), postpm (64. g) and prepm (.68 g). Seedling dry weight (per -seedling) was also greater at HM (.55 g) than at prepm (.64 g). Significant (P>.5) and positive correlations of seedling fresh weight with leaf number, area and leaf weight; root and shoot length; root and shoot fresh and dry weights; and shoot diameter were observed (Table ). Similarly, seedling dry weight was also correlated with those parameters. Seed maturation is a process of dry matter accumulation followed by changes in moisture content, size, germination, in addition to other physical, biochemi cal and morphological transformations. Seed moisture content is used to determine the beginning of seed filling (Dumolin et al., 994). In the present study, the seed moisture content declined gradually up to postpm stage of seed followed by a sharper decline at the subsequent stage, which is consistent with the observation of Parashiva and Dushyantha (4) in field bean and Muasya et al. (a) in common bean. Parashiva- Murthy and Kumar (4) stated that in field bean the seed moisture content was the highest (8.4%) at 5 days after anthesis (DAA), then gradually declined to 4.% at 6 DAA, while PM occurred at 5 DAA. In the current study, maximum viability and germinability were achieved at moisture content of.7% in HM seed which is well below the moisture content (6.9%) at physiological maturity (PM). This result is similar with the observation of Muasya et al. (b) in common bean who noted that maximum viability in seeds from all pod classes was achieved at moisture contents of -7%, which is well below the PM (58% moisture content) and closer to HM (% moisture content). Generally, matured seeds showed higher germination potential due to fully developed cotyledons and embryo. Maximum germination (8.%-84.55%) was recorded from the seeds harvested at PM up to HM, which is in agreement with Pokojska (999) in faba bean where maximum germination potential (95- %) was recorded at PM, 5-8 days after sowing and it continued until the last harvest date. Therefore, seeds from PM to HM maturity may be used as vegetable, bean sprout and/or dhal. Pokojska (999) again observed that maximum seed vigour was achieved at PM in faba bean. However, in the present study seed vigour was maximum TABLE : Effect of stage of seed maturity on dry mass content, seed germination, vigour, seedling weight, protein content and TSS in Lignosus bean (Trinidad genotype) (Expt. ) Stages of seed maturity Initial seed moisture Germination Seed vigour Seed protein Content Seed TSS Seedling weight (g/-seedlings) Fresh PrePM 6.75 a 5.4 c.79 d.5 b 7.65 d.68 c.64 c PM 6.9 a 8. b 55.8 b. c.7 c 7. b 8. b PostPM 6.8 a 8.59 ab 7.9 a.47 a.95 b 74.b 8. b HM.7b a 45. c.75 a.6 a 9.8 a.55 a In each column, figures bearing uncommon letter(s) differ significantly at P >.5 by DMRT, PM = Physiologically mature, HM = Harvest mature. Dry

6 Vol. 6, No. 5, 85 TABLE : Correlation between seedling weight and different seedling growth attributes in Lignosus bean (Trinidad genotype) (Expt. ) Dependent variable Independent variable r-value Mature leaf number.565** Leaf area.97** Leaf fresh weight.977** Leaf dry weight.958** Root length.48* Seedling fresh weight Shoot length.969** Root fresh weight.968** Root dry weight.944** Shoot fresh weight.98** Shoot dry weight.974** Shoot diameter.89** Mature leaf number.68** Leaf area.964** Leaf fresh weight.958** Leaf dry weight.989** Root length.498* Seedling dry weight Shoot length.964** Root fresh weight.949** Root dry weight.9** Shoot fresh weight.96** Shoot dry weight.987** Shoot diameter.84** N = 6 (4 seed maturity stages 4 replications); *, ** = Significant at P>.5 and P>., respectively. (7.9%) at postpm stage i.e., after PM. PM, postpm and H M produced more vi gorous seedling than PrePM which was reflected in leaf, root and shoot growth as indicated by correlation study (Table ). The PrePM stage of seed showed lower germination capability and vigour. This may have due to smaller dry matter accumulation in the seed that resulted in more decreased shoot and root growth than at the other stages of seed maturity. This agrees with the report of Avramiuc et al. (998) in broad bean who stated that reduction in seed germination and viabilities at different ages led to the decrease in seedling vigour index, mean shoot and rootlet length and weight. The seedling dry weight increased as seeds approached maturity and this is supported by the results of Chinnasamy and Bal () who investigated that in pea speed of germination, germination %, seedling length and dry weight increased as seed approached maturity. The significant and positive correlation of seedling weight (fresh and dry matter yield) with seedling growth parameters (number and weight of leaf, root and shoot length and weight) indicate that increased seedling growth would depend on better leaf production, shoot and root growth. It may be concluded that in Lignosus bean, physiological maturity and green harvestable seeds for vegetable were obtained at 4 days after flowering and seed quality at PM was also good. REFERENCES AOAC, (99). Assoc. Official An. Chemists., 5 ed Washington, D.C. Avramiuc, M., Strajeru, S., Fartais, L. and Savu, G. (998). Modification of some vigour indices in different age and viability seeds belonging to some maize and broad bean local populations. Cercetari Agronomice in Moldova, (/4): Chinnasamy, G. and Bal, A.K. (). The pattern of seed development and maturation in beach pea (Lathyrus maritimus). Can. J. Bot., 8(6): Das, S.S. (9). Studies on pod growth and seed quality in Lignosus (Dipogon lignosus) and country bean ( Lablab purpeureus). MS Thesis, Dept. Crop Bot., Bangladesh Agric. Univ., Mymensingh. Bangladesh. Dumoulin, V., Ney, B. and Eteve, G. (994). Variability of seed and plant development in Pea. Crop Sci., 4:

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