Floral morphology and seed yield in two Indigofera spp. as affected by shoot clipping

An experiment was carried out to know the effect of shoot clipping on floral morphology and seed yield in two Indigofera spp. viz. I. tinctoria L. and I. suffruticosa Mill. All parts of shoot including the main stem were clipped at 60 cm height from the base at 120 days after sowing (DAS). At 120 DAS, I. tinctoria already exhibited 1 flowering while I. suffruticosa showed no flowering. Shoot clipping had significant effect on almost all floral morphological features in two Indigofera spp. Flower raceme and pod raceme were fewer in clipped plant than in unclipped one. Shoot clipping altered the size of different floral parts to a great extent. The seed yield was decreased in clipped plant than that of unclipped one. Shoot clipping decreased flower and pod size and seed yield in two Indigofera spp.


Introduction
Indigofera is the third largest genus in legumes comprising ca. 750 species (Schrire, 2005) under the family Leguminosae and 19th (Mabberley, 1997) to 26th (Stevens, 2001) in size of all angiosperm genera. These species are grown in many countries of the world as ornamental, for production of indigo dye and also as herbal medicine (Ellison, 1999;Puri et al., 2007;Luiz-Ferreira et al., 2011). Indigofera tinctoria L. and I. suffruticosa Mill. are the two most important species frequently used to produce the indigo dye. Natural dyes are environmentally friendly and distinctive washing and sunlight fade-resistant than synthetic ones (Angelini et al., 1997).
Although the tale of indigo in this region (northern Bangladesh and parts of West Bengal) dates back to ancient times, Indigofera has been cultivated now only in small patches of Nilphamari district in Bangladesh as green manure, fuel crop, hedge plant, etc. (Anon., 2007). The resurgence of interest for natural dye to replace synthetic ones revives the cultivation probabilities of Indigofera for commercial dye manufacture in Bangladesh. Some research works have been conducted on different aspects of Indigofera production viz. morphological variations (Khan et al., 2008;Jahan et al., 2012); or to find out ways to increase biomass production by manipulating planting density and shoot clipping height (Rokonuzzaman, 2003), or by shoot clipping and Gibberellic acid application (Kabir, 2003). Previously, we reported the variation in phenology, floral and pollen morphology, and seed yield between two Indigofera spp. (Jahan et al., 2013). Clipping a portion of the shoot is one of the most important means for getting additional vegetative biomass yield and means of increasing plant growth (Costa et al., 1992;Rokonuzzaman, 2003;Howlader, 2013). The current research investigated the effect of shoot clipping on floral morphological features and seed yield in Indigofera spp.

Materials and Methods
The experiment was conducted at the Field, and Plant Morphology Laboratory of the Department of Crop Botany, Bangladesh Agricultural University, Mymensingh, during the period from April to December, 2011. The experiment was carried out following a randomized complete block design (RCBD) with three replications. The seeds of two Indigofera spp. viz. I. tinctoria and I. suffruticosa, were sown at 2-3 cm depth in the rows 30 cm apart. Emergence of seedling commenced at 4-5 days after sowing (DAS). Seedlings were thinned to maintain 15 cm distance between two adjacent seedlings. Different intercultural operations were done as and when necessary. It was found that shoot clipping at 60 cm height at 120 DAS resulted in higher biomass yield (Kabir, 2003;Rokonuzzaman, 2003). Therefore, the shoot clipping was done at 120 DAS where all the shoots of plants were clipped at 60 cm height from the base to maximize biomass yield for dye production. Final harvesting was done at 240 DAS.
Morphological features (colour, size and shape) of different floral parts (corolla, androecium and gynoecium), pod and seed were studied. The general morphological features of reproductive parts in two species are presented in Table 1. Quantitative data were recorded from randomly selected at least 5 individual plants of two species. Volume of the pod was determined following Khan et al. (2008). Collected data were statistically analyzed by using the computer software program MSTAT-C and the difference between means was adjudged by Duncan's New Multiple Range Test (DMRT).

Size of standard:
The size of standard significantly varied with species. It did not vary with shoot clipping. Interaction effect of species and shoot clipping for size of standard was significant. The standard was larger in I. tinctoria (18.82 mm 2 ) than in I. suffruticosa (13.16 mm 2 ) ( Table 2). Size of standard was the largest in clipped I. tinctoria (19.49 mm 2 ) and the lowest value was recorded in clipped I. suffruticosa (12.69 mm 2 ) ( Table 3). Size of wing: Size of wing significantly varied with shoot clipping and with species. The wing was larger in clipped plants (6.14 mm 2 ) than in unclipped plants (5.83 mm 2 ). The wing was larger in I. tinctoria (7.67 mm 2 ) than in I. suffruticosa (4.30 mm 2 ) ( Table 2). It was largest in clipped I. tinctoria (8.07 mm 2 ) and smallest in both unclipped and clipped I. suffruticosa (av. 4.30 mm 2 ) ( Table 3).

Size of keel:
Size of keel was greater in unclipped plant (16.31 mm 2 ) than in clipped plant (16.09 mm 2 ).
Length of stamen: Shoot clipping had no significant effect on length of stamen. There was significant difference in length of stamen between the two species. Stamen was longer in I. tinctoria (5.33 mm) than in I. suffruticosa (3.20 mm) ( Table 2). Interaction effect of clipping and species for length of stamen was significant. The stamen was the longest in both unclipped and clipped I. tinctoria (av. 5.33 mm) than in the other treatments (Table 3).

Length of carpel:
There was significant difference in length of carpel between shoot clipping treatments, and between the two Indigofera spp. The interaction effects of clipping and species are significant. Carpel had greater length in unclipped plant (4.73 mm) than in clipped plant (4.56 mm); and in I. tinctoria (5.32 mm) than in I. suffruticosa (3.97 mm) ( Table 2). The length of carpel was the greatest in unclipped I. tinctoria (5.47 mm) and the lowest in both unclipped and clipped I. suffruticosa (av. 3.98 mm) ( Table 3).

Length of ovary:
There was no significant difference in length of ovary between the two shoot clipping treatments. Length of ovary varied significantly with the species. Ovary was greater in I. tinctoria (4.02 mm) than in I. suffruticosa (3.10 mm) ( Table 2). Interaction effect of shoot clipping and species on length of ovary was significant. Ovary was the longest in both unclipped and clipped I. tinctoria (av. 4.02 mm) and the smallest in clipped I. suffruticosa (3.00 mm) ( Table 3).

Effect of shoot clipping on morphological features of pod and seed in two Indigofera spp.
Pod per raceme: Effects of shoot clipping and species on number of pod raceme −1 were significant. Pod raceme −1 was greater in unclipped one (18.16) than in clipped one (16.98) and in I. suffruticosa (24.43) than in I. tinctoria (10.71) ( Table 4). The interaction effect of shoot clipping and species on pod raceme −1 was significant. The pod raceme −1 was the greatest in unclipped I. suffruticosa (24.90) and the lowest in clipped I. tinctoria (10.00) ( Pod diameter: There was significant difference in pod diameter between the two Indigofera spp. Pod diameter was greater in I. suffruticosa (2.80 mm) than in I. tinctoria (2.57 mm) (Table 4). There was no significant difference in pod diameter between the shoot clipping treatments; interaction effect of shoot clipping and species was also significant (Tables 4 and 5).  In a column, figures bearing different letters differ significantly at P ≤ 0.05 by DMRT Pod volume: There was significant difference in pod volume between the shoot clipping treatments and also between the species. Pod volume was greater in clipped plant (115.85 mm 3 ) than in unclipped one (109.89 mm 3 ) and in I. tinctoria (139.00 mm 3 ) than I. suffruticosa (86.74 mm 3 ) (Table 4). Interaction effect of clipping and species for pod volume was significant. Clipped I. tinctoria showed highest pod volume (147.00 mm 3 ) and both unclipped and clipped I. suffruticosa showed the lowest pod volume (av. 86.75 mm 3 ) (Tables 4 and 5).

Seed per pod:
There was no significant difference in seed pod −1 between the shoot clipping treatments (Table 4). Significant difference in seed pod −1 was also found between the two Indigofera species. The interaction effect of shoot clipping and species for seed pod −1 was also significant. Seed pod −1 was greater in I. tinctoria (7.25) than in I. suffruticosa (4.17) ( Table 4). It was the greatest in both unclipped and clipped I. tinctoria (av. 7.25) and the lowest in both unclipped and clipped I. suffruticosa (av. 4.18) ( Table 5).
Seed size: Shoot clipping and species had significant effects on seed size. Seed size was greater in clipped plant (2.80 mm 2 ) than in unclipped plant (2.70 mm 2 ) and in I. tinctoria (3.33 mm 2 ) than in I. suffruticosa (2.19 mm 2 ) ( Table 4). The interaction effect of shoot clipping and species for seed size was significant. Seed size was the highest in both unclipped and clipped I. tinctoria (av. 3.34 mm 2 ) and the lowest in both unclipped and clipped I. suffruticosa (av. 2.20 mm 2 ) ( Table 5).

1000-seed weight:
There was no significant difference in 1000-seed weight between the two shoot clipping treatments (Table 4). Thousand seed weight was also significantly different between the two species. The interaction effect of shoot clipping and species for 1000-seed weight was also significant. Indigofera tinctoria (5.62 g) had greater 1000-seed weight than in I. suffruticosa (3.64 g) (Table 4). Thousand seed weight was higher in clipped I. tinctoria (5.70 g) than clipped I. suffruticosa (3.58 g) ( Table 5).
Seed yield: Seed yield significantly varied with shoot clipping and with species. The interaction effect of shoot clipping and species for seed yield was also significant. Seed yield was greater in unclipped plant (13.17 g plant −1 ) than in clipped one (8.78 g plant −1 ) ( Table 4) and in I. tinctoria (14.70 g plant −1 ) than in I. suffruticosa (7.20 g plant −1 ) ( Table 4). Seed yield was the highest in unclipped I. tinctoria (18.67 g plant −1 ) and the lowest in clipped I. suffruticosa (6.75 g plant −1 ) ( Table 5).

Discussion
Shoot clipping had adverse effect on floral morphology in the both Indigofera spp. (Tables 2 and 3), although it increases biomass production in indigo plant (Kabir, 2003;Rokonuzzaman, 2003). Shoot clipping results in loss of leaves, the source of supplying assimilates to sink-flowers, developing pods and seeds in legumes (Hossain et al., 2006a,b;Mondal et al., 2011). Shoot clipping may, therefore, influence TDM production, and seed and biomass yield through assimilate production and its partitioning into different parts depending on the magnitude of source loss (Hossain et al., 2006a,b;Mondal et al., 2011). The clipped plants showed fewer flower raceme −1 than in unclipped plant (Tables 2 and 3). Shoot clipping also altered the size of different floral parts to a great extent, and had an adverse effect on seed yield in indigo (Tables 4 and 5). Yield is the function of number of flowers raceme −1 x number of pods raceme −1 x number of seeds pod −1 x 1000-seed weight (Mondal et al., 2011). The clipped plants exhibited fewer flower raceme −1 which resulted in fewer pods raceme −1 and finally lower seed yield (g plant −1 ) in clipped indigo plants. In cowpea, partial source removal induces increase in pod and seed yield (Hossain et al., 2006a,b) through the production of higher flowers plant −1 with reduced rate of floral abscission (Hossain et al., 2006b). In the present study, shoot clipping was done during and/or around first flowering (Jahan et al., 2013). Therefore, the flowering was delayed and seed formation was also delayed leading to a reduced seed yield in clipped plants in two Indigofera spp. Moreover, the shoot clipping treatments invariably cause a reduction in the amount of biomass partitioned to the reproductive organs, and affects flowering (Gutman et al., 2001). Leaf clipping at flower initiation stage induces the capacity to compensate source loss through the re-growth of leaves in soybean (Board and Harville, 1998;Borras et al., 2004). Higher rate of photosynthesis in remaining leaves of partially clipped plants compared to the leaves in intact plants (Rao and Ghildiyal, 1985) may contribute the compensation to source loss by clipping. However, leaf clipping did not affect seed yield in high yielding mungbean genotypes while it reduced yield in low yielding one (Fakir, M.S.A., personal observation).
The variations in floral morphology and seed yield exist between the species (Tables 3 and 5; Jahan et al., 2012). Shoot clipping had more adverse effect in I. tinctoria especially on seed yield compared to in I. suffruticosa (Table 5). The variations between the species in response to clipping might came from the difference in growth rate between the species (Jahan et al., 2013). The pods raceme -1 was significantly smaller in I. tinctoria than in I. suffruticosa both in clipping and control conditions, although shoot clipping increases fruit setting percentage in both the species (Tables 3 and 5). At 120 DAS when clipping treatment was done, I. tinctoria already exhibited 1 st flowering while I. suffruticosa showed no flowering (Jahan et al., 2013). Therefore, clipping in this experiment produced fewer flowers and pods and finally lowered the seed yield in I. tinctoria.
It can be concluded that shoot clipping adversely affected size of flower and pod and seed yield in both of the two Indigofera spp.