The results of present investigation were briefly discussed as the ultra-structure of the pod and seed coats of the susceptible varieties of ML 267 and MGG 295 as against pre-harvest sprouting, the pod wall exhibited deep cracks and witnessed a greater number of pores on its surface (Fig 1 A and B). The thickness of podwall was lesser than K 851 and LGG 450. The podwall thickness of MGG 295 was 423 µm. The fairly large, elongated and a greater number of functional and turgid trichomes with high density on podwall surface. The outer surface of pod wall got thin cuticular layer followed by one celled epidermis. These made easy access for movement of water when it was wet or subjected to simulated rainfall (Fig 2 A and B). Water droplets adhere to trichomes could easily absorb water. Thus, wetting of seed coat or absorption of water could also be enhanced due to the high density of trichomes on the outer surface of the podwall.
The movement of water could also be made easy due to thin cuticular layer and one - celled epidermis and kept moistened. Further, the mesocarp and endocarp seen in perforated tissues exhibited large longitudinal cracks in MGG 295 as well as in ML 267. This is immediately followed by a large locular space (0.48 mm) in MGG 295 and 1.06 mm in ML 267 formed a reservoir around the seed proper for incoming water. Several researchers (
Dougherty and Boerma, 1984 in soybean;
King and Richards, 1984 in wheat;
Lush and Evans 1980 in mungbean) revealed that pod morphological characters are made congenial for wetting, rapid movement and water absorption of podwall and kept it moist condition which leads to pre-harvest sprouting under adverse climatic conditions due to thin podwall ultrastructural features like deep cracks with more number of pores, thin unicellular cuticular layer, high density, size and shape of the trichomes on the pod wall got thin cuticular layer followed by one celled epidermis. K 851 and LGG 450 have shown pre-harvest sprouting resistance due to attribution of pod morphological characters. LGG 450 and K 851 pod wall surface had no cracks with very few numbers of pores (Slight and slits). Trichomes sparsely with short, weiry twisted few in number along the margin seen as it dried and less turgid nature of trichomes and thick podwall. A very few short, flattened, less turgid, wiry and twisted trichomes (Fig 3 A and B) in these genotypes indicate their dysfunction for absorption of water. This further emphasizes less scope for trapping of water droplets when they are wet. These morphological characters shown resistance against pre-harvest sprouting. Similar findings were also reported by
Singh et al., (2017), Sarfraz Ahmad et al., (2014), Satyanarayana et al., (1991) and
Harris (1987).
The seed coat of ML 267 and MGG 295 (Fig 4A and B) are relatively thinner 77.3 µm (MGG 295) than that of K 851. The cotyledons of ML 267 had smaller starch granules embedded within protein bodies. The above described layers of pod wall, locular space and seed coat of ML 267 and MGG 295 clearly indicate an easy access to water movement across the seed layers. All the aforesaid structural features culminated for easy movement of imbibed water and might prone the seed of these genotypes to pre-mature sprouting, when they were subjected to simulated or unseasonal rainfall.
The thickness of the mesocarp and endocarp of LGG 450 together measures 195 µm (Fig 5A and B). The locular space was reduced conspicuously in LGG 450 while it was very small compared to that of K 851. Both these genotypes had three times lesser locular space compared to that of ML 267 (Fig 5A). The locular space at placental region or at other places ranges from 34-39 µm in LGG 450. The seed coat of LGG 450 and K 851 is thicker (Fig 6 A and B) compared to ML 267 and MGG 295 (77.3 µm). The cotyledons contain few but larger starch granules, partially covered with protein deposits. The embryo was seen interlocked within cotyledons measuring 2 x 3 and 558 x 275 µm. The embryonic space around the embryo was relatively more at mid width. This indicates that the embryo is unready to take up imbibitional growth for sprouting unlike that of ML 267 (Fig 7A). The longitudinal embryo (Fig 7B) was seen surrounded by a large space; embryonal space also indicates a space for accumulation of water helped at the time of imbibition.
The thicker mesocarp almost tight with endocarp together with narrow locular space (no gap in case of LGG 450) forms a barrier for impeding free movement of water or with less scope for accumulating water around the seed coat. The cotyledons contain almost naked starch granules with splashed protein bodies indicate less prone for absorption of water; also, the median width of the embryo indicates its unreadiness for sprouting. These observations in the testing genotypes are in accordance with the findings
of
Sarfraz Ahmad et al., (2014) and
Harris (1987).
The matured seed germination on pod itself under high rainfall conditions triggered with absorption of water from the podwall layers across the seed coat into embryo. The movement of water from podwall across its layers via locular space and seed coat into embryo depends on the rate, pace and pattern of movement of water. The quantity of water with time that could reach the embryo trigger imbibition, hydration of embryo, hydrolysis of reserves and biochemical process of imbibed seed which decides the seed whether sprout, delay or not to sprout at all. Thereby, the path of water movement across the tissues from seed coat to embryo was traced using iodine water, as per the technique developed by
Mc Donald (1994). The study conducted with selective genotypes (ML 267, MGG 295, LGG 450 and K 851) based on their response of sprouting to simulated rainfall, revealed that the genotype ML 267 sprouted early while LGG 450 failed to sprout. Under conditions of wetting / simulated rain, the water movement took its path across podwall, locular space, podwall, space around cotyledons and embryo as clearly seen in ML 267 (Plate 1a). The time-course movement of water in the seed was traced by using I2-KI treated water showing dark areas (Plate 1a). This path had been found dissipating slowly first into the locular space within 4 h slightly around cotyledon
i.e., in the podwall space and by 12 h, prominent accumulation of water occurred in locular space indicating intense accumulation of water all through the circumference of podwall (Plate 1b), whereas in LGG 450 which exhibited relative tolerance to wetting, showed a slow and irregular diffusion of water through the podwall and locular space by 6 h (Plate 1b) but a prominent dark colour could be seen by 6 h, 12 h in the locular space and around cotyledons, (partially) in Plate 1c. By 24 h, much accumulation of water was seen only in the locular space with a little diffusion into the cotyledonary area. A white embryo scar indicates no reach of water to the embryo (Plate 1d) even by 24 h. The restricted water movement may also be attributed to very low sprouting compared to the variety ML 267. Water imbibition (%) by pod showed highly significant and positive correlation with seed germination percentage. Higher amount of water absorbed by the pod makes sufficient moisture available for the seeds present inside to initiate process of germination. Similar role of pre-harvest with rate of water imbibitions through pod wall has been reported by
Uwins et al., (1996), Sarfraz Ahmad (2014),
Renata Anna et al., (2016) and
Singh et al., (2017).
The genotype ML 267 showed dark colouration in the podwall layers indicates the presence of water between the podwall and seed coat moved carrying I2 ions. The colour intensity was still more in the locular space, which represents the presence of more water accumulated around the seed coat. The intensity of colour was lighter in the seed coat wall but darker in the space between seed coat and cotyledons. The cotyledonary area showed a little shade, indicating the presence of relatively less water. The embryonal space also showed dark colour clearly suggesting the presence of water around the embryo (Fig 7A and B). It was concluded that water accumulation inevitably occurs in locular space around the seed and embryo which was readily prone the seed for sprouting condition. The genotype LGG 450 showed dark colouration in the podwall layers or seed coat, even though they were faint to dark colouration in very tight locular space. It was suggested that I
2 water movement in this seed was very slow and low even after 6 h of treatment with I2 water. The cotyledons also showed very faint colouration at the distal end.
The variation in anatomical/ultra-structural features of pods and seeds of mungbean varieties of ML 267, MGG 295, LGG 450 and K 851 examined under the scanning electron microscopy revealed that more number of pores with few deep cracks on podwall surface, large elongated and a greater number of turgid and functional trichomes, thin unicellular cuticular layer with one-celled epidermis, a large locular space and protein bodies embedded between many smaller starch granules in cotyledonary area. ML 267 and MGG 295 have shown the above traits which favors for susceptibility to pre-harvest sprouting. In a time course study, the quantity of water accumulated, rate of moisture absorption, speed, path and pattern of water movement across the podwall and seed coat including locular space, cotyledonary area and embryo shown that ML 267 and MGG 295 accumulated more water in turn sprouted early. Such accumulation of water, absorption and movement of water across the podwall and seed coat were less in LGG 450 and K 851 due to their morphological features. Thereby, it was either delayed or no sprouting in LGG 450 and K 851 repectively. Determination of susceptibility/tolerance as against pre-harvest sprouting in mungbean genotypes (ML 267, MGG 295, LGG 450 and K 851), based on the traits of topography, architectural built up, nature of podwall surface and thickness, type of cells of podwall and seed coat including locular space. The biochemical nature and stimulating enzymes of cotyledons were led to sprouting. The variation in above characters would be useful for crop improvement programme and development of tolerance/ resistance in mungbean varieties against pre-harvest sprouting. Ultra-structural studies in understanding and determining the factors for pre-harvest sprouting of seeds in various crops are available. However, majority of the scanning electronic microscopy studies are in the lines of understanding the physiological biochemistry of pre-harvest sprouting. In a study by
Cai and Chen (2008), it was reported that the activities of a-amylase, IAA, GA1+3 and ZR were higher in easy-germinated rice than in uneasy-germinated rice. However, ABA content was lower in easy-germinated rice than in uneasy-germinated ones. The research findings further indicate that pre-harvest sprouting in rice is related to a-amylase, endogenous hormone contents and glume structure. However, research on ultra-structural studies using Scanning Electronic Microscopy in determining factors for pre-harvest sprouting in mungbean is scanty.
In a study using SEM in wheat, it was observed that starch granules in sprouted seed samples were partially hydrolyzed. Further, the high-performance size exclusion chromatography (HPSEC) profiles indicated that the starch of sprouted samples had relatively lower molecular weight than that of non-sprouted samples. Overall, the results indicated that a-amylase activity caused changes to the physicochemical properties of the pre-harvest sprouting damaged wheat
(Simsek et al., 2014).
Fresh seed dormancy is a desirable trait in mungbeanto overcome the problem of pre-harvest sprouting. Earlier research on fresh seed dormancy in mungbean indicated developing cultivars with a fresh seed dormancy of 10 to 15 days could curtail the losses associated with pre-harvest sprouting
(Lamichaney et al., 2017). The biochemical activities that determine the fresh seed germination (FSG) and high pre-harvest sprouting in mungbean genotypes also are to be investigated for reducing the yield losses. It is established that high activity of a-amylase is responsible for high fresh seed germination and pre-harvest sprouting. For rapid identification of genotypes with varied levels of fresh seed dormancy and pre-harvest sprouting, a-amylase can be an effective biochemical marker
(Lamichaney et al., 2017).
Despite identification of mungbean genotypes with high degree resistance to pre-harvest sprouting based on desirable plant, seed, pod traits, ultra-structural studies and biochemical activities such as low a-amylase activity, it is necessitated to thoroughly screen the genotypes at field level. Several researchers have evaluated the mungbean germplasm at field level for establishing the pre-harvest sprouting tolerance (
Singh et al., 2017). For field resistance to pre-harvest sprouting, availability of suitable donor germplasm lines in breeding programmes is mandatory.
Recent research findings in other crops also suggest the application of genetic tools in imparting pre-harvest sprouting resistance. For example, in wheat, genetic loci that determine pre-harvest sprouting resistance were identified that facilitate breeding for sprout-resistant wheat cultivars. These results on molecular approaches not only provide genetic resources for pre-harvest sprouting resistance, but also the important breeding tools for marker-assisted selection based on genome-wide linkage mapping for pre-harvest sprouting using 15K SNP arrays (
Lingli li et al., 2021).
