Correlative Influence of Axes on Senescence of Cotyledons Following Germination of Mung Bean Vigna Radiata (L.) Wilczek Seeds

Research Article

Austin J Plant Biol. 2015;1(1): 1005.

Correlative Influence of Axes on Senescence of Cotyledons Following Germination of Mung Bean Vigna Radiata (L.) Wilczek Seeds

Pal L and Kar RK*

Department of Botany, Visva Bharati University, India

*Corresponding author: Kar RK, Plant Physiology and Biochemistry Laboratory, Department of Botany, Visva Bharati University, Santiniketan 731235, West Bengal, India

Received: May 05, 2015; Accepted: June 24, 2015; Published: June 30, 2015


Immediately after seed germination cotyledons undergo transient development culminating in senescence, which is different from that of a leaf. In the present study senescence of mung bean (Vigna radiata) cotyledons following germination has been characterized to understand correlative influence of axes. Cotyledons isolated from axes showed retardation of storage protein degradation as well as increased chlorophyll synthesis followed by senescenceinduced decline. When one of the pair of cotyledons was detached from the axis, the attached cotyledon showed same characters like intact cotyledons but the detached one showed the behavior similar to the isolated cotyledons. When epicotyls were removed after 3 days incubation cotyledons remained attached with axes for longer period and decline in chlorophyll content retarded compared to control, but decline in protein level remained unaffected. Removal of root tip marginally delayed the decline in both chlorophyll and protein level. Morphological studies also showed maintenance of greenness and fullness of cotyledons when isolated or epicotyls removed. Histological studies revealed that storage bodies started degenerating following germination from the peripheral zone while chloroplast development progressed around vascular bundle ending with signs of senescence in the last phase. Both storage body degeneration and chloroplastic senescence was delayed in case of isolated cotyledons. Therefore, cotyledons in case of epigeous germination of seeds pass through complex developmental changes. Immediately after germination storage mobilization starts from the periphery to provide soluble substrates to growing axes along with photosynthetic development around vascular bundle; the latter ends with senescence-induced degeneration influenced by emerging epicotyl acting possibly as sink.

Keywords: Chlorophyll; cotyledon senescence; epicotyl decapitation; protein; root tip removal


Senescence is a programmed developmental process that occurs at every stage of plant’s life cycle. Thus following seed germination seedling development starts with cotyledon(s) undergoing transient development along with the growth of embryonic axes followed by senescence in case of most dicotyledonous seeds. Indeed cotyledons have a short life span, at the end of which these finally shrink and detached from the axes with the appearance of differentiated leaves [1]. The major physiological function of cotyledons is to provide substrates through storage mobilization for the development of the growing seedling until differentiation of photosynthetically efficient leaves. In dicot seeds, particularly non-endospermic legume seeds storage proteins remain sequestered compactly in protein storage vacuoles of parenchyma cells of cotyledons [2] and during germination such proteins undergo hydrolysis by proteinases resulting in production of amino acids that mobilizes to the embryonic axes to support growth [3]. During this period, cotyledons gradually become green in colour and plastids develop into functional chloroplasts [4] before these finally senesce and detached from axes. Fundamentally senescence process in leaves and cotyledons may be same, but as a special organ cotyledons are likely to have a differently regulated senescence under the correlative influence of growing embryonic axes during germination.In the present study attempt has been made to characterize cotyledon senescence of Vigna radiata seeds in the context of correlative influence of growing axes following germination by monitoring chlorophyll and protein levels and morphological and histological studies of cotyledons either in isolation or under removal of epicotyl or root tip.

Materials and Methods


Seeds of Vigna radiata (L.) Wilczek (var. B1) were collected from Pulses and Oilseeds Research Station, Berhampur, Murshidabad, West Bengal. Healthy seeds were first surface sterilized with 1% sodium hypochlorite solution for 5 minutes, rinsed in distilled water several times and dried on tissue paper. Seeds were then placed in covered plastic Petri dishes (9 cm) on Whatman no.1 filter paper soaked in distilled water. These sets were then incubated in darkness under controlled temperature (30±2°C) in a seed germinator. After 24 h germination, germinated seeds or isolated cotyledons were incubated either in light or in darkness for different durations and at intervals cotyledons were analysed for chlorophyll and protein contents. Cotyledons from seedlings with root tip removed or decapitated epicotyl were also used for similar assessments. Assessments for chlorophyll and protein contents were done at 2 day intervals in most of the cases except in case of comparative assessment between attached and detached cotyledons and in case of root tip removed cotyledons where samples were collected at 1 day intervals.

Chlorophyll estimation

Cotyledons (5 pairs in number) were washed with distilled water and the surface water was soaked with tissue paper. These were then homogenized with 5 ml 80% acetone and centrifuged at 5000×g for 10 minutes. The supernatants were decanted and made up to a definite volume (5 ml). The contents of chlorophyll a and chlorophyll b separately as well as total chlorophyll content were estimated by recording the absorbance at 663nm and 645nm in a spectrophotometer following the method of Arnon [5]. The chlorophyll content was expressed as μg/pair of cotyledons.

Protein estimation

For the determination of the protein content, the residues (pellet) of the samples, from which the chlorophyll was removed, were digested in 2 ml of 1(N) NaOH for 1 h at 800C. After digestion the extract was diluted appropriately with distilled water. To 1 ml of this final (diluted) extract, 1 ml of the mixture of Reagent a 10% sodium carbonate in 0.5 (N) NaOH, 1% copper sulphate and 2% sodium potassium tartarate in the ratio of 20:1:1 was added. After 5 min Folin Phenol Reagent (Reagent B), diluted to 1:2 with water, was added to the mixture and the protein content was estimated by measuring the absorbance of the blue colour developed after the reaction at 650nm in a spectrophotometer according to the method of Lowry et al. [6]. A standard curve was prepared using Bovine Serum Albumin (BSA). The protein content was expressed as mg/pair of cotyledons.

Morphology and histological studies of cotyledons

Morphology of cotyledons for seedlings subjected to epicotyl decapitation, root tip removal, cotyledons isolated from or left attached to axes was studied and photographed. Anatomy of cotyledons was studied by observing transverse sections of cotyledons, either intact or isolated, under light microscope at different intervals during incubation in light. Starch grains and protein body distribution were determined by staining freehand sections of cotyledons with iodine and toluidine blue, respectively. All the sections used in these experiments were obtained by cutting single cotyledons transversely. Live sections were stained with 0.01% toluidine blue in 0.1% aqueous sodium tetraborate (for protein bodies) and 0.5% iodine in 5% aqueous potassium iodide (for starch grains) for light microscope observations.


It was observed that the intact cotyledons remained attached with the germinating axis until day 5 after which they were detached from the axis. Total chlorophyll content of these cotyledons incubated in light increased from day 1 up to day 3 followed by a decrease. On the other hand, isolated cotyledons were maintained up to day 13 and the chlorophyll content of cotyledons constantly increased from day 1 to day 7 reaching a value almost 5 times of the maximum level recorded in intact cotyledons and then decreased gradually thereafter up to day 13 under light (Figure 1c). Although the content of chlorophyll a of the cotyledons (both intact and isolated) was far more than that of chlorophyll b, the trend of changes with incubation was same for both chlorophyll a and chlorophyll b contents (Figures 1a and 1b). There was negligible chlorophyll production in dark conditions. As there was no significant chlorophyll production noted in dark in case of subsequent experiments, no data were presented for chlorophyll level changes in cotyledons from seeds incubated in dark. But protein content of cotyledons was checked in both light and dark conditions to ascertain any difference in protein level changes, mainly due to storage mobilization in case of light-incubated and dark-incubated seedlings.However, the protein content was found to decrease drastically from day 1 to day 5 to a minimum level in intact cotyledons similarly under both light and dark condition, whereas the protein content was maintained more or less at the same level under both light and dark incubation in isolated cotyledons after an initial fall at day 3 (Figure 1d).

In case of another experiment, where one cotyledon of the pair of cotyledons from an intact seedling was detached while other one left attached with axis, cotyledons were analysed for contents of total chlorophyll and chlorophyll a and b separately incubating seeds in light only and protein content during incubation both in light and darkness (Figures 2 and 3). Both the contents of chlorophyll (a and b and total) (Figures 2a, b and c) and protein (Figures 3a and b) of the cotyledons which were attached with the germinating axis, declined similar to the intact cotyledons i.e. an initial increase up to day 3 followed by decline in case of chlorophyll a and b and a gradual decline from the beginning for protein content both in light (Figure 3a) and dark (Figure 3b). On the other hand, the detached cotyledons (without axes) showed the characters like isolated cotyledons i.e. gradual increase in the contents of chlorophyll a and b (up to day 5) and retarded decline of protein level.