The structure and size of the orchid seed are among the most striking characteristics of the Orchidaceae. Orchid seeds are minute, weigh from 0.3 to 14 µg and measure from 0.250 to 1.2 mm in length and 0.090 to 0.270 mm in width [4]. This was followed by structural analyses in which the fine balloon-shaped seed coats could be clarified as helical wall thickening by oxygen ion etching. An overview of the functional morphology of the dust-like seeds appeared shortly after [14]. As the seeds of orchids are the smallest among the seeds produced by flowering plants, it is difficult to study their structural details with an ordinary optical microscope [5]. The reason for this is the fact that the embryo occupies a very small portion of the seed volume. As a result, even relatively large increases in embryo volume do not appreciably decrease the percent air space of the seed. Seed and embryo length and embryo width appear to be independent of each other. However seed width could be affected by increases in embryo girth. Hence, seed width may not be as valid an independent parameter as seed length. Our findings indicate that seed morphometry can be a useful taxonomic tool. However, judgement should be tempered by the realization that age and environmental conditions may affect measurements [5]. The seed surface morphology of 82 species of orchids are analyzed and illustrated. The value of seed morphology assessing the relationship of different taxa of Orchidaceae is emphasized. They have made Orchid seed into 8 groups and made a dichotomous key up to groups [8]. Orchid seeds are unique. They differ from those of most angiosperms and resemble the so-called ‘dust seeds’ of other plants. The seed air space (SAS) plays a key role in the distribution of orchid species and the large SAS is beneficial in increasing the dispersal area [2].

            Studies on seed morphometry of 10 epiphytic orchids reported highest seed volume in Cymbidium bicolor (11.80 mm³ × 10^-3) and lowest volume in Liparis elliptica (0.22 mm³ × 10^-3). Highest number of testa cells in Coelogyne breviscapa (10.76) and the lowest number in Bulbophyllum mysorense (1.87). Highest embryo volume in Coelogyne breviscapa (2.26 mm³ × 10^-3) and lowest embryo volume in Aerides maculosum (0.075 mm³ × 10^-3). Air space in more in Aerides maculosum (79.79 %) and less air space in Liparis elliptica (17.07 %) (Krishna swamy et al., 2004) [10]. [9] in his studies on seed morphometry of Dendrobium species recorded six species (D. aquem, D.herbaceum, D. heterocarpum, D. lawianum, D. macrostachyum, D. nutans) from Karnataka with yellow to cream yellow color seeds. Highest seed volume recorded in D. lawianum (3.26 mm³ × 10^-3) and lowest seed volume in D. nutans (0.811 mm³ × 10^-3). Highest number of testa cells recorded in D. lawianum and lowest number in 3.2 D. heterocarpum. Highest embryo volume in D. nutans (0.585 mm³ × 10^-3) and lowest embryo volume in D. macrostayum (0.18 mm³ × 10^-3). Highest air space reported in D. heterocarpum (91.05%) and lowest in D. macrostaychum (22.40%). [13] Conducted 10 Seed morphometric studies from Eastern and Western Ghats of India. The colour of the seeds in all investigated species was pale yellow to yellow and light brown to white. The highest seed volume was observed in Calanthe triplicata (2.44 mm3×10-3) followed by M. densiflora, O. arnottiana, O. ensiformis, followed by L. zeylanica. In V. testaceae and V. tessellata, the seeds were of lesser volume and small sized. Whereas minimum L/W ratio was observed in A. praemorsa (2.67).  The L/W ratio in other investigated taxa was, 4.87 in V. testaceae, 3.79 in A. rigida, 3.39 in L. zeylanica, 3.31 in O. ensiformis, and 2.77 in V. testellata. Seeds with higher ratio of seed volume/embryo volume (more than 2.2) especially in C. triplicata, V. tessellata, and V. testacea are expected to be more buoyant than those with a lower ratio of seed volume/embryo volume.  These are widely spread-out species in Western Ghats of South India. A higher percentage of airspace was also noticed in these orchid taxa. In general, the dust-like minute seeds are suitable for long distance dispersal by wind.

            An a symbiotic method for orchid seed germination was developed in 1921. After Knudson’s media B and C were formulated, orchids growing and hybridization became widespread. Hybrids which early growers may not have even imagined became possible reported by Yam & Arditti [17] in their work History of orchid propagation: a mirror of the history (2009).  [15-16] recorded some Seed morphometric studies using light microscope of 10 species from Manipur. Highest seed volume is observed in Cymbidium iridioides (15372.8 µm³ × 10³) followed by Paphiopedilum hirsutissimum (8506.9 µm³ × 10³), while minimum seed volume was observed in Taeniophyllum glandulosum (159.3 µm³ × 10³). Species like Vanda coerulea, Hygrochilus parishii, Arides odorata and Rhynchostylis retusa have lesser seed volumes with 281.7 µm³ × 10³, 416.5 µm³ × 10³, 422.1 µm³ × 10³ and 479.4 µm³ × 10³ respectively. Maximum embryo length is observed in Paphiopedilum hirsutissimum (312.5 µm) and minimum in Taeniophyllum glandulosum (85.0 µm). Maximum embryo width is observed in Paphiopedilum hirsutissimum (135.0 µm) and minimum in Taeniophyllum glandulosum (42.5 µm). Maximum embryo volume is observed in Paphiopedilum hirsutissimum (3069 µm3 × 103) and minimum in Taeniophyllum glandulosum (82.4 µm3 × 103). Maximum sir space is found in Cymbidium irididides (14134.5 µm³ × 10³) and minimum in Rhynchostylis retusa (32.02 µm³ × 10³). [15] [16] reported seven threatened Himalayan orchids (listed in Appendix II of CITES) belonging to three different subfamilies, and exhibiting terrestrial (Arundina graminifolia, Cypripedium cordigerum, Habenaria marginata, Herminium monorchis), mycoheterotrophic (Gastrodia falconeri) or epiphytic (Aerides multiflora, Pholidota articulata) life modes. Seeds were fusiform, filiform or ovoid in shape and their size showed a direct correlation with plant habit. Cypripedium cordigerum has the highest seed volume (4.26) and lowest seed volume Aerides multiflora (0.088). Arundina graminifolia records highest embryo volume (0.71) and lowest embryo volume in Pholidota articulate (0.020). Air space in more in Gastrodia falconeri (91.69 %) and less air space in Arundina graminifolia (49.93 %). Most commonly the testa cells are tetragonal, hexagonal or polygonal; sometimes the cell shape is irregular. We distinguish three kinds of testa cell shape: (1) all cells are more or less isodiametric (regardless of their actual shape); (2) the cells are elongate in the longitudinal axis of the seed (prosenchymatic) and rectangular; and (3) the cells are elongate but rounded at the ends [6]. [7] stated that the increase of embryo volume (EV) results in increasing seed weight, thus improving the ability of the seed to float on air and the distribution of the species. These species showed remarkable variation in seed color (off-white, brown and yellow) and shape (fusiform, spathulate and filiform). Seven species were found to have elongate seeds, while the remaining species had truncate seeds. Seeds with the highest percentage of air space were found in H. gibsonii (84.91 ± 11.34%) followed by H. frucifera (84.77 ± 9.45%). The number of testa cells was found to be more or less constant at the genus level, and with straight, sinuous, or undulate walls. H. diphylla has highest seed volume (16.97 ± 2.14) and lowest seed volume in H. brachyphylla (1.26 ± 0.50). H. diphylla has highest embryo volume (16.29 ± 4. 36) and lowest embryo volume in H. crinifera (0.45 ± 0.08) [7].

Material  and Methods

The mature seeds were collected from the naturally dehiscing capsules from the natural habitats of different species from North Karnataka. Then color of the seeds were noted by tallying with the color codes by POWO KEW color barcode [11]. The seeds were washed in 70% alcohol and dried for the measurement process.

            Dried Seeds were spread on carbon stub, were gold plated and photographed under JEOL 100 CXII ASID 4d model scanning electron microscope at 10-20kv. Measured the seed length and breadth in micrometers and nanometers. Testa cell length and breadth is also measured.

Seed volume is calculated using the Arditti et al., 1979

                                    Seed Volume= 2{(W/2)²(L/2)100}

            The embryos are prolate shaped. The embryo are of various colors from light yellow to green to brownish to orange. The measurement of embryo is done through light microscope, measuring its length and breadth of the embryo. As embryo are elliptical in cross section, their volume is calculated with the Arditti et al., 1979

                                    Embryo volume= 4/3 (1.047) (L/2) (W/2)²

Air space is calculated using the above formula

                                    Air space= (Seed volume-Embryo volume/Seed volume) 100

Results:

Seed color

            Conducted 106 different species of seeds were collected and washed with 70% alcohol to remove some fibers and threads. Than seeds were observed under stereo-microscope to record the colors using POWO KEW color bar code [11]. Different color seeds were recorded (CY-Cream Yellow, GY-Golden Yellow, DB-Dark Brown, W-White, Y-Yellow, B-Brown, BY-Brown Yellow, GW-Greenish White, GY-Green yellow, DY- Dark Yellow, TY-Transparent Yellow, CW-Cream White, GB-Greyish Black, CB-Chocolate brown, LB-Light Brown) (Table 1).

            [13] recorded color of the seeds was pale yellow to yellow and light brown to white. Malaxis densiflora (White), Oberonia ensiformis (Light yellow), Acampe praemorosa (Light brown), Luisia zeylanica (Yellow), Vanda testacea (Light yellow), Vanda tessellata (Yellow) from eastern and Western Ghats. Prashant (2007) [12] in his work reported color of seed ranges from the pale yellow to cream yellow and brown to golden brown from Shivmogga district. [7] reported Seed color ranges from off-white, pale yellow to yellow and pale brown to dark brown. Testa cells are usually transparent from Western Ghats of India in the year 2016. Krishna swamy et al.,  [9] reported D. aqueum (Yellow), D. herbaceum (Cream yellow), D. heterocarpum (Yellow), D. lawianum (Yellow), D. macrostachyum (Yellow), D. nutans (Yellow) from Karnataka in the year 2007. Krishna Swamy et al., [10] reported pale yellow to yellow, brown and white from some epiphytic orchid from Karnatak in the year 2004. Aerides maculosum (Brown), Coelogyne breviscapa (Light yellow), Cymbidium bicolor (Yellow), Oberonia ensiformis (Pale Yellow), Pholidota pallida (Pale Yellow), Vanda parviflora (Brown) from his investigation.

Shape of Seed:

            The shape of orchid seeds varies and could be ellipsoid, oblongoid, ovoid, globose, filamentous, spindle, irregular, fusiform, or filiform. But further apart from this using Kew Bulletin glossary 2023 gives various shapes for seeds. The shapes are as follows: FOO- Fusiform with opening one side; TOO- Turbinate with opening one side; OOO- Obovoid with opening one side; POO- Prolate with opening one side; SEOO- Spindle Elongated with opening one side; FFOO- Fusiform Flat with opening one side; FDOO- Fusiform Dentate with opening one side; OCB- Obovate Close at both ends; FLCB- Fusiform Linear Close at both ends; SEOO- Spindle Elongated with opening one side; OBOO- Oblong with opening one side; FEOO- Fusiform Elongated with opening one side; OVOO- Ovid with opening one side; OEOO- Oblong Ensiform with opening one side; TTOO- Turbinate Twisted with opening one side; FHOO- Fusiform Hooks with opening one side; LTO- Lanceolate with two side opening; OTO- Oblong with Two side Opening; OTOO- Obconical Twisted with opening one side; LFTO- Linear Fusiform with Two Side Opening; OFTO- Obconical Flat with Two side Opening; FTTO- Fusiform Twisted with Two side Opening; UTO- Urceolate with Two side Opening; FTOO- Fusiform Twisted with Opening one side; UTOO- Urceolate Twisted with One Side Opening; FLTOO- Fusiform Linear Twisted Opening One side; UFTO- Urceolate Flat Opening  One side; ULTO- Urceolate Linear with Two Side Opening; ULOO- Urceolate Linear with Opening One side; OBVOO- Obovate Opening One side; OHOO- Obconical with Hooks Opening one side; POO- Pyriform with Opening One side; OBVOO-Obovate Opening at One side; UOO- Urceolate Opening at One side; OBTOO- Obovoid Twisted Opening at One side; EOO- Elliptic Opening on One side; PTOO- Prolate Twisted Opening on One side; FLTOO- Fusiform Linear Twisted with Opening on One side; OVOO- Ovoid Opening on One side; ULOO- Urceolate Linear Opening on One side; FLOB- Fusiform Linear Opening at both sides; FTFOO- Fusiform Twisted Flat Opening on One side; OBOO- Obconical with Opening One side; OTLOO- Obconical Twisted Linear Opening on One side;  PFTOO- Prolate Flat Twisted Opening on One side; TTOO- Turbinate Twisted Opening on One side; PFTOO- Prolate Flat Twisted on Opening One side; FTHOO- Fusiform Twisted Hooks with opening one side; FTHCE- Fusiform Twisted Hooks with close ends; LLOO-Linear Lanceolate Opening one side; FLOO- Fusiform Linear Opening one side (Figures 1-17).

            [13] reported M. densiflora seeds were quadrilateral-shaped with blunt ends. O. ensiformis seeds were spindle shaped but with a bulged central part. A. praemorsa were ovoid or spathulate shape. V. tessellata and V. testacea were spindle shaped or oblong. Prashant (2007) [12] in his work reported seeds of Acampe praemorsa are long broadly ellipsoid in shape. Aerides crispum and Aerides ringens vary in their shape from crescent shape to the broadly ellipsoidal and ridges are close and looks like twisted rope. Cymbidium aloifolium the seeds are long, transparent, spindle shape. Dendrobium crepidatum is more or less long and elongated with blunt ends. Habenaria heyneana and Habenaria longicorniculata seeds are long, fusiform and transparent. Luisia zeylanica is more or less long elongated and curved. Trias stocksii are long elongated curved with thin and flake like pointed ends. Seeds of Vanda tessellata are spindle shape with blunt ends. Vanda testacea are spindle shaped with flat side and a pointed end. [7] reported spathulate in H. brachyphylla, H. grandifloriformis, H. roxburghii, H. suaveolens and H. rariflora. Fusiform in H. commelinifolia, H. crinifera, H. heyneana, H. longicorniculata, H. longicornu, H. marginata and H. plantaginea. Filiform in H. digitata, H. foetida, H. furcifera, H. gibsonii and H. ovalifolia. Krishna swamy et al., [9] reported D. aqueum (long and elongated), D. herbaceum (oval to spindle), D. heterocarpum (elongated with bulged central part), D. lawianum (elongated with bulged central part), D. macrostachyum (spindle), D. nutans (spindle) from Karnataka in the year 2007.

Seed volume:

            Highest seed volume is present in some species like Acampae ochreaceae (1.16), Cymbidium bicolor (2.06), Didymoplexsis pallens (3.0), Eulophia nuda (6.47), Eulophia pratensis (4.2), Eulophia picta (8.37), Habenaria furcifera (3.55), Habenaria multicaudata (3.53), Habenaria rariflori (2.26), Nervilia concolor (2.21), Oberonia verticiliata (1.13), Peristylus plantagenius (2.21), Zeuxine reflexa (3.5). Lowest is seen in Bulbophyllum sterile (0.04), Cottonia pedencularis (0.05), Oberonia bicronis (0.03), Oberonia falconeri (0.04), Oberonia recurva (0.08) (Table 1).

            [13] reported M. densiflora seed volume is 0.355 mm³×10³. O. ensiformis seed volume is 0.443 mm³×10³. A. praemorsa seed volume is 0.203 mm³×10³. V. tessellata seed volume is 0.230 mm³×10³ and V. testacea has seed volume 0.298 mm³×10³.

            Prashant (2007) [12] in his work reported highest seed volume is noticed in Cymbidium aloifolium (1.671 mm³×10³), it is followed by seeds of Habenaria longicornculata (5.2207 mm³×10³), Habenaria heyneana (3.6154 mm³×10³) and Dendrobium crepidatum (1.2207 mm³×10³). The rest of the taxa Acampe praemorsa (0.2265 mm³×10³), Aerides crispum (0.4372 mm³×10³), Aerides ringens (0.1914 mm³×10³), Luisia zeylanica (0.2527 mm³×10³), Polystachya flavescens (0.2258 mm³×10³), Trias stocksii (0.3210 mm³×10³), Vanda tessellata (0.3829 mm³×10³) and Vanda testacea (0.2210 mm³×10³) have seeds of lesser volume and smaller in size.

            [7] reported H. diphylla has highest seed volume (16.97 ± 2.14 mm³×10³) next is H. roxburghii (7.90 ± 1.24 mm³×10³). Lowest seed volume is H. crinifera (0.86 ± 0.12 mm³×10³) next is H. brachyphylla (1.26 ± 0.50 mm³×10³).

            Krishna swamy et al., [9] reported D. aqueum (0.464 mm³×10³), D. herbaceum (0.487 mm³×10³), D. heterocarpum (0.236 mm³×10³), D. lawianum (1.49 mm³×10³), D. macrostachyum (0.181 mm³×10³), D. nutans (0.586 mm³×10³) from Karnataka in the year 2007.

Embryo Volume:      

            Embryo analysis is done in stero and light microscope to check record the parameter like length and breadth. Embryo is prolate to ovid shape. Highest embryo volume is Coelogyne imbricate (0.4), Crepidium verscicolor (0.5), Cymbidium bicolor (0.63), Habenaria grandifloriformis (0.63), Habenaria longiconiculata (2.0), Habenaria plantaginaea (2.0), Nervilia concolor (1.35). Lowest embryo volume is seen in many species like Oberonia mucrunata (0.006), Oberonia bicornis (0.009), Luisia macrantha (0.008) (Table 2).

[13] reported highest embryo volume in V. tessellata (4.26 mm³×10³)and next is M. densiflora (2.58 mm³×10³). Lowest embryo volume is in O. ensiformis (1.31 mm³×10³) and next is A. praemorsa (2.08 mm³×10³).

            Prashant (2007) [12] in his work reported highest embryo volume in Habenaria longicornuculata (1.99 mm³×10³), it is followed by Habenaria heyneana (0.95 mm³x10³). Lowest embryo volume in Aerides ringens (0.12 mm³×10³) and Acampae praemorsa (0.16 mm³×10³).

            [7] reported H. digitata has highest embryo volume (16.29 ± 4. 36 mm³×10³ mm³×10³) next is H. rouxburghii (6.60 ± 2.12 mm³×10³). Lowest embryo volume is H. crinifera (0.45 ± 0.08 mm³×10³) and H. gibsonnnii (0.40 ± 0.09 mm³×10³). Embryo color reported is brown to yellow color. Krishna swamy et al., [9] reported D. aqueum (0.46 mm³×10³), D. herbaceum (0.48 mm³×10³), D. heterocarpum (0.23 mm³×10³), D. lawianum (1.49 mm³×10³), D. macrostachyum (0.18 mm³×10³), D. nutans (0.58 mm³×10³) from Karnataka in the year 2007. Embryo color varies from yellow to pale yellow.

After Seed volume and embryo volume, calculated the air space, which is very important for germination process.

Air space

Highest air space is in Acamape ocheraceae (97.37%), Cheriostylis flabellata (97.15%), Malaxis densiflora (98.49 %), Oberonia ensiformis (96.11%), Habenaria heyneana (86.70%). Air space is highest in many genera. Lowest in Dendorbium barbatulum (7%) (Table 2). 

            [13] reported the highest air space percentage in Vanda tessellata (62 %)and next is Malaxis densiflora (60.50 %). Lowest air space percentage is Acampe praemorsa (35.53 %) and next is Oberonia ensiformis (38.19 %). [12] in his work reported highest air space percentage in Cymbidium aliofolium (93.4 %), it is followed by Habenaria heyneana (7.69 %). Lowest embryo volume in Vanda tesellata (12.0 %) and Vanda testacea (15.61 %). [7] reported H. digitata has the highest air space percentage (81.56 ±11.23 %) and next is H. foetida (76.21 ± 9.59 %). Lowest seed volume to embryo volume is H. grandifloriformis (0.86 ± 0.46 %) and H. diphylla (4.00 ± 2.56 %).  [7] reported seed volume to embryo volume ratio in D. aqueum (51.70 %), D. herbaceum (47.54 %), D. heterocarpum (91.05 %), D. lawianum (54.16 %), D. macrostachyum (22.40 %), D. nutans (29.96 %) from Karnataka in the year 2007.

Acknowledgement

Authors are mainly thankful to USIC DST SAIF Scanning Electron Microscope analysis, Karnatak University Dharwad for SEM photographs and measurements. Authors are thankful to Karnatak University’s Research Student fellowship (URS) for conducting this research programme.

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