Assessment of somatic embryogenesis potency in Indian soybean [Glycine max (L.) Merr.] cultivars
Thankaraj Salammal Mariashibu1, Kondeti Subramanyam, Muthukrishnan Arun, Jeevaraj Theboral,Manoharan Rajesh, Sampath Kasthuri Rengan1, Rajan Chakravarthy1,
Markandan Manickavasagam& Andy Ganapathi*
1Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, India
Received 10 October 2012; revised 22 July 2013
Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication reports the development of somatic embryogenesis in a liquid culture medium from immature cotyledons of G. max.
Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic embryos and percentage of explants that responded were higher with 45.24 µM 2,4-D. The proliferation of somatic embryos for three successive cycles was achieved in 22.62 µM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 µM) indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L- asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with 74.42% of response.
Keywords: Amino acids, Genotype, Immature cotyledon, Somatic embryogenesis, Soybean
The soybean (Glycine max [L] Merrill) is a species of legume native to East Asia, widely grown for its edible bean which has numerous uses. The plant is classed as an oilseed rather than a pulse by the Food and Agricultural Organization (FAO). The USA, Argentina, Brazil, China, and India are the world's largest soybean producers and represent more than 90% of global soybean production. India produces 10.12 million metric tons per year and is the fifth largest producer in the world1. Soybeans contain significant levels of omega-3-fatty acids, isoflavones, and phytic acid which play an important role in human health. The use of meat-based diets among the growing world’s population has also increased the demand for soybean protein for livestock and poultry feed. Soybean cultivation in India was negligible until
1970, but it grew rapidly and surpassed over 6 million tons in 2003. In India, more than 70 cultivars are presently considered to be most promising as germplasm. However, soybean cultivation is threatened by various biotic and abiotic factors. In India, the average yield is approximately one ton per hectare, which is lower than the international average of two tons per hectare. The existing cultivars should be improved genetically to increase the yield without extending the cultivation area. However, traditional breeding practices have led to a limited success due to the narrow genetic variation and the presence of barriers to genetic crosses. During the past decade, considerable achievements have been made in the field of plant genomic research, which has helped in the identification and cloning of genes controlling desirable plant traits. However, the availability of successful regeneration protocol is a prerequisite for the transfer of desirable gene(s) into soybean to improve this crop plant.
The induction of somatic embryogenesis for in vitro plant regeneration provides several advantages over the traditional organogenesis2. Somatic embryogenesis provides an excellent morphogenetic
______________
*Correspondent author Telephone: +91 431 2407086 Fax: +91 431 2407045, 240702
E-mail: aganapathi2003@rediffmail.com subramanyamkondeti@gmail.com
Present address: 1Temasek Life Sciences Laboratory Limited, 1 Research Link, National University of Singapore, Singapore 117604, Singapore.
First two authors (TSM and KS) have contributed equally.
system for investigating the cellular and molecular process underlying differentiation3. In addition, somatic embryogenesis also provides the possibility to produce artificial seeds and valuable tools for genetic engineering and germplasm conservation via cryopreservation4,5. Somatic embryogenesis was first reported in soybean by Christienson et al6. The majority of soybean somatic embryogenesis protocols are based on the use of immature cotyledons as the explant7-13. Even though there are reports on somatic embryogenesis on soybean, only a limited number of cultivars can be induced to produce somatic embryos.
In fact, most research on somatic embryogenesis of soybean has been restricted to the highly regenerative cultivar like Jack and Williams 82. Nevertheless, somatic embryogenesis from immature cotyledons is highly genotype dependent, and some genotypes are recalcitrant in nature9,14-17. Majority of the Indian soybean cultivars are also recalcitrant to somatic embryogenesis; to date, there is no information on the specific somatic embryogenesis of Indian cultivars.
Hence, the present investigation undertaken to standardise a rapid and reliable protocol for somatic embryogenesis in Indian soybean cultivars and to study the influence of the genotype on somatic embryo induction. In addition to this we compared the effect of solid and liquid medium on somatic embryogenesis of Indian soybean cultivars.
Materials and Methods
Explant source and preparation—Indian soybean cultivar Pusa 16, a commercially important cultivar in India, was preferred for standardizing somatic embryogenesis. Plants were grown in an experimental garden at the Department of Biotechnology, Bharathidasan University, Tiruchirappalli (10°40'58.9469''N; 78°44'28.608''E) between October and February. The date of flowering was marked by tagging, and immature pods were collected 15 days after anthesis. These pods, containing immature cotyledons of 2–7 mm in length, were surface sterilised by immersion in 70% (v/v) ethanol for 30 sec followed by 0.1% (w/v) HgCl2 for 5 min; the pods were then washed thrice with sterile distilled water. The immature seeds were aseptically collected from the pods, and the embryonic axis was cut away and discarded. The resulting immature cotyledon halves were used as explants to induce somatic embryogenesis.
Somatic embryo induction—The embryogenic potential of the immature cotyledon explants was tested using solid somatic embryo induction medium
(SSEIM) containing Finer and Nagasava lite (FNL) macro salts8, Murashige and Skoogs (MS) micro salts18, B5 vitamins19, 87.64 mM sucrose, 2,4 dichlorophenoxy acetic acid [2,4-D (13.57–361.92 µM)], and naphthalene acetic acid [NAA (16.11–80.55 µM)]; the pH of the medium was adjusted to 5.6–5.8 before solidifying with 0.2% phytagel. A pair of cotyledons was cultured in a culture tube (15 × 150 mm) containing 10 mL of solid medium by placing the adaxial side of the cotyledons on the SSEIM medium. In case of liquid somatic embryo induction medium (LSEIM), 10 immature cotyledon explants were cultured on an orbital shaker (Orbitek, Chennai, Tamil Nadu, India) at 100 rpm in a 150 mL Erlenmeyer flask containing 35 mL of LSEIM, composed of the following: FNL macro salts, MS micro salts, B5 vitamins, 29.21 mM sucrose, 2,4-D (13.57–
361.92 µM), and NAA (16.11–80.55 µM). The media were autoclaved at 121 °C for 15 min. All of the cultures were incubated at 25±2 °C with a 23 h photoperiod at a light intensity of 5–10 µEm−2s−1.
Somatic embryo proliferation—Somatic embryo proliferation was performed in liquid somatic embryo proliferation medium (LPM) containing FNL macro salts, MS micro salts, B5 vitamins, 29.21 mM sucrose with different concentrations of 2,4-D (13.57–180.96 µM), and NAA (5.37–53.70 µM). Fifty globular-shaped somatic embryos were transferred into flasks containing LPM and maintained as above for the somatic embryo induction. After two weeks of culture in this proliferative medium, 50 globular embryos were again transferred to fresh LPM for further proliferation. This proliferation process was continued for up to three cycles.
Histodifferentiation and maturation of somatic embryo—Early-stage globular embryos from LPM were transferred to 35 mL of liquid histodifferentiation medium (LHM) containing FNL macro salts, MS micro salts, B5 vitamins, 87.64 mM sucrose with different concentrations of NAA (5.37–53.70 µM) for the differentiation of globular into cotyledonary stage embryos. After differentiation in LHM, green-coloured cotyledonary-staged embryos were transferred to liquid maturation medium (LMM) containing FNL macro salts, MS micro salts, B5 vitamins, 164 mM D-sorbitol, and 87.64 mM sucrose and cultured for two weeks. All the cultures were incubated on an orbital shaker at 100 rpm at 25±2 °C with a 23 h photoperiod at a light intensity of 5–10 µEm−2s−1.
Desiccation, germination, and acclimatization—
The fully matured cream-coloured cotyledonary stage
embryos were desiccated by the air-drying method of Parrott et al20. Cream-coloured, well-matured embryos (25) were transferred to sterile empty petri plates (120 mm diameter) and sealed with parafilm.
Humidity was maintained by placing 1 cm3 MS basal medium near the embryos. The petri plates were placed in the dark for 0–7 days at 25±2 °C.
The desiccated embryos were transferred to MS basal semi-solid medium for conversion into plantlets.
The germinated embryos were transferred to small pots containing vermiculite, sand, and soil (2:1:1).
The plantlets were covered with polythene bags to maintain a high humidity (80%). After one week of hardening, the bags were removed gradually, and the plantlets were transferred to earthen pots.
Effect of amino acids on somatic embryo induction, proliferation, and histodifferentiation—Amino acids, such as L-alanine (10.0–80.0 mM), L-asparagine (1.0–20.0 mM) and L-glutamine (3.0–40.0 mM), were tested for any role in the somatic embryo induction, proliferation and histodifferentiation. The immature cotyledon explants were inoculated into LSEIM containing different concentrations of different amino acids. Each treatment comprised of fifty explants in replicates of five. The number of embryos induced was counted after 3 weeks of culturing. In the similar way globular embryos were inoculated into LPM and LHM containing different concentrations of different amino acids. Proliferated and histodiffrentiated embryos were counted after 2 weeks of culturing.
Genotypic effects on somatic embryogenesis—After standardising the media composition, growth regulator requirements and supplements (amino acids) for somatic embryo induction using cultivar Pusa 16, immature cotyledon explants from seventy Indian cultivars were cultured in LSEIM to test their somatic embryo induction response with reference to their genotypic features. The genotypes tested were as follows: ADT−1, Alankar, Ankur, Birsa soy 1, Bragg, DS 228, Co 1, Co Soya 2, DS 97−12, Durga, Gaurav, Gujarat soybean 1, Gujarat soybean 2, Hardee, Hara soy, Indira soy 9, Improved pelican, Palam Soya, JS 2, JS 71−05, JS 75−46, JS 76−205, JS 79−81, JS 80−21, JS 90−41, JS 93−05, JS 335, Kalitur, KB−79, KHSb 2, Lee, Lsb 1, MACS 13, MACS 57, MACS 58, MACS 124, MACS 450, MAUS 1, MAUS 2, MAUS 32, MAUS 47, MAUS 61, MAUS 61−2, MAUS 71, MAUS 81, PK 416, Pusa 20, Pusa 22, Pusa 40, Punjab 1, RAUS 5, PK 262, PK 308, PK 327, VL Soya 1, VL Soya 21, PK 471, PK 472, PK 564, PS 1024, PS 1029,
PS 1042, PS 1092, PS 1347, Punjab 1, Pusa 16, Pusa 24, Pusa 37, NRC 12, and NRC 37.
Histology and photomicrography—For histological studies, immature cotyledon explants inoculated in LSEIM at various time intervals, proliferating embryogenic clumps, and embryos at different developmental stages were fixed in formalin, acetic acid, and 50% ethyl alcohol (0.5:0.5:90, v/v/v) for 48 h and then dehydrated through graded series of ethyl alcohol and tertiary butyl alcohol and were finally embedded in paraffin (58–60 °C). Serial sections of 8 µm thickness were cut with a rotary microtome (2035 BIOCUT, Germany), stained in Toludene blue orange and observed under bright field microscope. Organization and other features of cells and tissues were photomicrographed using Nikon optihit microscope and Nikon sterio microscope with photographic unit Nikon FX – 35 camera (Nikon, Japan).
Results and Discussion
Explant age and size—Immature cotyledon explants that were 2–5 mm in size (most of these explants were collected between 12–15 days after anthesis) (Fig. 1a) responded well to the somatic embryo induction and produced more embryos than larger (>6 mm) and older explants; the latter did not respond well to somatic embryo induction and produced non-embryogenic calli in both liquid and solid somatic embryo-induction medium. Immature cotyledons from field-grown soybean plants are more suitable explants for somatic embryogenesis21,7. Indeed, Lazzeri et al.22 have reported that the size and age of immature cotyledon explants are crucial factors for somatic embryo induction in soybean.
Effect of growth regulators on somatic embryo induction—The immature soybean cotyledons started to produce small green-coloured protuberances from the margin and abaxial surfaces after five weeks of culture on SSEIM, whereas the immature cotyledons placed in LSEIM begin to produce such protuberances (Fig. 1b) after 14 dyas (2 weeks) of culture. Within 21 days (3 weeks), the protuberances developed into globular stage embryos (Fig. 1d), which were clearly visible in LSEIM. The induced globular somatic embryos started to detach from the explant after 25 days culture (Fig. 1e). The rate of response and the number of somatic embryos varied significantly with the physical nature of medium: the liquid medium (LSEIM) produced better results than the solid medium (SSEIM) (Table 1). Further, the type of auxins in the medium played a vital role in the
induction of somatic embryos from immature cotyledon explants. The auxins, 2,4-D and NAA, were tested at different concentrations to assess their efficiency in somatic embryo induction. Although both auxins induced somatic embryogenesis, 2,4-D produced better results than NAA in terms of the percentage of response and the number of somatic embryos per explant, where 45.24 µM 2,4-D in LSEIM resulted a 52.43% response and an average of 5.23 globular somatic embryos per cotyledon. NAA produced an average of 2.83 globular stage somatic embryos and a 17.81% response at 26.85 µM in LSEIM (Table 1). The somatic embryos induced on 2,4-D were friable, translucent, yellowish-green in colour and globular- to torpedo-shaped (Fig. 1d, e, and f). In contrast, the somatic embryos induced on NAA were compact, opaque and pale green in colour, with an advanced morphology, forming cotyledon-like structures. Somatic embryos induced by NAA exhibited normal morphology and started to differentiate into the cotyledonary stage and produced adventitious roots before attaining physiological maturity.
The auxin type, concentration, and exposure time are important for the initiation of somatic embryogenesis in legumes20. Previous studies have demonstrated that either 2,4-D or NAA was required to induce somatic embryos from immature cotyledon explants, and most of these research articles have recommended the use of 181 µM 2,4-D in solid medium to induce somatic embryogenesis16,21,23-27
. In the present study also 180.96 µM 2,4-D evoked somatic embryo induction in a solid medium (SSEIM). However, a lower concentration, 45.24 µM, of 2,4-D in LSEIM produced a higher number of somatic embryos with a higher percentage of response. Several studies have also included NAA at lower concentrations20,22,28,29
, but in the present study a lower number of somatic embryos with NAA was observed. It is interesting to note that the embryo induction from explants required only 2–3 weeks in LSEIM, whereas induction in the solid medium required 5−6 weeks26. It seems likely that the liquid medium allowed a better distribution of the nutrients, which may be an important factor for embryogenic tissue, in which there is a significant competition for nutrients. Hence, the liquid based medium may provide better selection regime during the transgenic somatic embryo recovery30. Effect of growth regulator on embryo proliferation—To increase the number of somatic embryos, the globular stage embryos were separated from the explants and sub-cultured in LPM containing
Table 1Effect of 2,4-D and NAA on somatic embryo induction from immature cotyledon explants of soybean cv. Pusa16 in solid
and liquid medium
[Values are mean ± SE from 5 independent experiments]
Percentage of response
Mean no. of globular embryos/cotyledon*
Hormone con (µM)
SSEIM LSEIM SSEIM LSEIM
Control NR NR ND ND
2,4-D
13.57 07.81±0.2i 20.23±0.6d 1.24±0.04k 2.06±0.03g 22,62 10.64±0.2f 37.66±0.7b 1.92±0.04g 3.64±0.02c 45.24 11.65±0.1e 52.43±1.0a 2.54±0.03d 5.23±0.02a 90.48 17.43±0.3b 21.26±0.4c 2.83±0.04c 4.26±0.03b 180.96 27.27±0.5a 15.65±0.3f 3.65±0.02a 3.04±0.02d 271.44 13.26±0.1c 13.20±0.3i 3.15±0.01b 2.34±0.01f 361.92 12.83±0.2d 10.05±0.2k 2.03±0.02e 1.42±0.01j NAA
16.11 05.82±0.2m 14.23±0.3h 1.00±0.02m 1.40±0.01k 26.85 06.63±0.2l 17.81±0.3e 1.41±0.02i 2.83±0.02e 37.59 07.41±0.4j 15.25±0.2g 1.62±0.03h 2.00±0.01h 53.70 08.52±0.4g 11.43±0.2j 2.01±0.04f 1.83±0.01i 64.44 08.23±0.2h 09.67±0.2l 1.26±0.02j 1.23±0.01l 80.55 07.24±0.3k 07.62±0.1m 1.02±0.02l 0.83±0.01m SSEIM–Solid somatic embryo induction medium; LSEIM–Liquid somatic embryo induction medium; NR–No response; ND–Not determined due to no response. *Results were recorded after 21 days (3 weeks) of culturing in case of LSEIM and after 5 weeks in case of SSEIM. Each treatment comprised of 50 explants in replicates of five. Percentage of response = No. of immature cotyledons responded for somatic embryo induction/Total No. of immature cotyledons cultured X 100. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5% level.
Fig. 1—Somatic embryogenesis from immature cotyledon explant of Indian soybean cv. Pusa 16. a: Immature cotyledon explants cultured in LSEIM containing 45.24 µM 2,4-D, 20 mM L-glutamine, and 29.21 mM sucrose (bar = 1.0 mm), b: Somatic embryo induction from edges of immature cotyledon explant in LSEIM containing 45.24 µM 2,4-D, 20 mM L-glutamine, and 29.21 mM sucrose after 12 days of culture (bar = 1.0 mm), c:
Immature cotyledon explant with embryos after 14 days (2 weeks) of culture (bar = 1.0 mm), d: Globular embryos in LSEIM containing 45.24 µM 2,4-D, 20 mM L-glutamine, and 29.21 mM sucrose after 21 days (3 weeks) of culture (bar = 1.0 mm), e:
Globular embryos separated out of explant in LSEIM containing 45.24 µM 2,4-D, 20 mM L-glutamine, and 29.21 mM sucrose after 25 days of culture (bar = 1.0 mm), f: Proliferated embryogenic clumps showing globular embryos in LPM containing 22.62 µM 2,4-D, 10 mM L-asparagine, and 29.21 mM sucrose (bar = 1.0 mm), g: Histodifferentiated somatic embryos in LHM containing 10.74 µM NAA, 30 mM L-glutamine, and 87.64 mM sucrose. h: Matured somatic embryos in LMM containing 164 mM D-sorbitol and 87.64 mM sucrose, i:
Desiccated somatic embryos, j: Germinated somatic embryo in MS basal medium, k: Hardened soybean plantlet, l: Acclimatised soybean plant surviving in greenhouse.
various concentrations of either 2,4-D or NAA. The LPM without auxins did not show any proliferation.
The medium containing 2,4-D promoted the proliferation (Fig. 1f) within two weeks and also an increase in the size of the somatic embryos. A highest number of globular stage embryos was observed with the use of 2,4-D at 22.62 µM (Table 2), a proliferation trend that was observed for up to two successive cycles, although increasing the concentration of 2,4-D did not yield any further positive effect. The proliferation experiments conducted using NAA failed to show a positive response, indeed it favoured the conversion of globular embryos to cotyledonary stage embryos (unpublished data not shown).
The proliferation of somatic embryos has a great advantage in transgenic recovery. Therefore, it is desirable to have secondary embryo proliferation from primary embryos. Embryo initiation in presence of 2,4-D is preferable for the maintenance of proliferation in embryogenic soybean cultures24, and a higher level of 2,4-D apparently prevents the development and maturation of the embryo while still allowing proliferation7,21. However, some reports have indicated that a high level of 2,4-D (82.4 µM) was required for the multiplication of somatic embryos on solid medium31, whereas a low level of 2,4-D (20.6 µM) was beneficial in liquid culture8,9. In agreement with the latter, the present observations indicated the requirement of a lower concentration of 2,4-D in liquid medium for the proliferation of somatic embryos.
Somatic embryo histodifferentiation and maturation—It was observed in the present study that the proliferated somatic embryos need to be transferred to the medium with NAA or without growth regulators for histodifferentiation. However, the somatic embryos induced with NAA did not require any change in medium, and a simple sub-culture in medium with the same composition favoured histodifferentiation. The globular embryos initiated from immature cotyledon explants and the proliferating early-staged embryos cultured with 2,4-D progressed through all of the stages of development (globular–heart–torpedo) and reached the cotyledonary stage (Fig. 1g) when cultured in histodifferentiation medium containing NAA. The liquid histodifferentiation medium (LHM) without plant growth regulator also exhibited differentiation (Table 3). The lack of a requirement of growth regulators for the differentiation into the cotyledonary stage is in agreement with previous studies, where it has been suggested that the globular embryos may be capable of producing their own hormones to support early development8,32. However, the differentiation rate (37.43 embryos per 50 embryos) and quality of the embryos were significantly higher in the histodifferentiation medium containing 10.74 µM NAA, where 72.05% of the globular stage embryos progressed to the cotyledonary stage. The somatic embryos differentiated on NAA showed advanced stages of embryo morphology, when compared with the embryos differentiated on basal medium. The cotyledonary stage embryos differentiated in presence
Table 2Effect of 2,4-D on proliferation of globular embryos initiated from immature cotyledon explants of soybean cv. Pusa 16 in LSEIM containing 45.24 µM 2,4-D, and 29.21 mM sucrose
[Values are mean ± SE from 5 independent experiments]
2,4-D conc (µM)
Response (%)
Mean no. of globular embryos in first cycle of
proliferationA
Mean no. of globular embryos in second cycle
of proliferationB
Mean no. of globular embryos in third cycle
of proliferationC
13.57 70.21±2.0c 076.42±2.0e 75.86±1.6e 73.42±1.2e
22.62 76.83±2.2a 101.63±2.2a 99.02±1.4a 96.45±1.8a
45.24 72.46±1.9b 090.41±2.4b 91.06±2.1b 88.04±1.8b
90.48 68.47±1.4d 087.64±2.1c 85.82±1.9c 85.02±1.6c
135.72 60.08±1.6e 081.83±1.9d 80.46±1.8d 79.23±1.9d
180.96 41.65±0.9f 070.27±1.8f 69.65±1.6f 68.84±1.8f
AFifty fresh globular stage embryos were inoculated per treatment in replicates of five. BFifty embryos from the first cycle of proliferation were sub-cultured in fresh medium. CFifty embryos from second cycle proliferation were sub cultured in fresh medium. Percentage of response = No. of embryos responded for first proliferation cycle/Total No. of embryos cultured X 100. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5% level.
of NAA had expanded cotyledons of a compact nature and exhibited typical bipolarity with a distinct radical and hypocotyl (Fig. 1g) whereas, the embryos differentiated in the basal medium displayed narrow cotyledons and were light green in colour. Somatic embryo differentiation in LHM was faster than the basal medium and required only two weeks to reach the cotyledonary stage. Further maturation of the somatic embryos in liquid medium was achieved without any growth regulator, and the somatic embryos lost their green colour when they reached maturation and were ready for germination. In the present study, the histodifferentiation and maturation process required 4 weeks.
Effect of amino acids on somatic embryo induction, proliferation and histodifferentiation—Amino acids are most commonly used as a nitrogen source33. Nitrogen is indispensable for somatic embryogenesis since it is required as key components in plant structure, functions and for building blocks such as proteins, nucleic acids, and plant hormones. Amino acids, including L-alanine, L-asparagine, and L-glutamine, were tested for their role in somatic embryo induction, proliferation, and histodifferentiation. Although all the three amino acids showed positive response to somatic embryo induction, L-glutamine at 20 mM was found to be optimum with maximum percentage of response (74.42%) and mean number of somatic embryos (8.45 embryos per cotyledon). Among the three amino acids tested for somatic embryo proliferation, L-asparagine at a concentration of 10 mM favoured the somatic embryo
proliferation with 85.64% of response and produced 170.24 globular somatic embryos. The highest percentage of response (80.82%) to somatic embryo histodifferentiation and mean number of cotyledonary stage embryos (56.62) was noticed when the LHM supplemented with 30 mM concentration of L-glutamine. It has been proved that, the amino acids, such as L-asparagine and L-glutamine, played a positive role in the development of soybean embryogenic cell suspensions8,34. It is established beyond doubt that addition of L-glutamine during embryo development increased the size of the somatic embryos35,36. Formation of embryogenic clumps was improved in cell suspensions of soybean when L-asparagine was added to the culture medium37. Loganathan et al.38 have observed a doubling of the embryogenic response when L-glutamine was added to the somatic embryo induction medium. The present study also indicated the positive influence of L-glutamine and L-asparagine in somatic embryo development.
Desiccation, germination, and acclimatization—
The embryos matured (Fig. 1h) in liquid maturation medium did not germinate into plantlets without desiccation. The differentiated embryos were desiccated for different period of time (1−7 days).
During the desiccation process, the embryos lost their water content, shrivelled up and reached physiological maturity (Fig. 1i). Somatic embryos desiccated for 5 days showed a higher germination response (40.88 %), whereas non-desiccated embryos failed to germinate (data not shown). Embryos with well-defined shoot apices germinated within 15 days (Fig. 1j), whereas embryos lacking a defined shoot apex required another two weeks for germination. The plants germinated from somatic embryos were transferred to plastic cups (Fig. 1k) containing sand:soil:vermiculite (2:1:1 v/v/v). After the emergence of a new leaf, the plantlets were transplanted into pots and acclimatized in greenhouse (Fig. 1l).
Maturation and germination is a significant rate- limiting factor in most somatic embryogenesis studies. In the present study, germination was also a rate-limiting factor, thus, the somatic embryos were subjected to an air-drying desiccation treatment to enhance the germination. Indeed, it has been reported that a physiological state permitting germination can be rapidly induced by desiccating the somatic embryo in a dry, sterile petri dish for a week20, Jang et al.37
Table 3Effect of NAA on histodifferentiation of globular embryos to cotyledonary stage in LHM containing 87.64 mM sucrose [Values are mean ± SE from 5 independent experiments]
NAA conc (µM)
Response (%)
Mean no. of cotyledonary stage embryo
0.00 46.04±1.2g 27.04±0.8c
5.37 68.26±1.4c 30.62±0.8b
10.74 72.05±1.6a 37.43±0.9a
16.11 65.83±1.3e 26.67±0.6d
26.85 70.65±1.8b 25.21±0.5e
37.59 67.41±1.2d 23.06±0.3f
53.70 57.26±1.1f 19.23±0.2g
Each treatment comprised of 50 globular embryos in replicates of five. Mean values of five independent experiments (±) with standard errors. Percentage of response = No. of globular embryos converted into cotyledonary stage embryos/Total no. of globular embryos cultured X 100. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5% level.
have recorded 90% germination of somatic embryos after 4 days of desiccation. An air-drying method, in which somatic embryos were desiccated in an empty sealed petri dish for 3–5 days, has also been reported to have resulted in the best germination efficiency among four tested methods: fast, slow, air, and KCl methods26.
Genotypic effects on somatic embryogenesis—The liquid-based somatic embryo induction medium (FNL macro salts, MS micro salts, B5 vitamins, 45.24 µM 2,4-D, 20 mM L-glutamine and 29.21 mM sucrose) developed in the present study was employed to screen 71 Indian soybean cultivars. Six Indian cultivars, namely Pusa 16, DS 97−12, Gujarat soybean 1, PK 416, VL soya 1, and PK 472 responded favourably, though, with varied efficiency (Table 4).
Among these, Pusa 16 showed a higher response rate (74.42%), with an average of 8.45 embryos per immature cotyledon explant in the liquid system. The other Indian cultivars did not respond well or produced fewer numbers of somatic embryos with less percentage of response (unpublished).
Parrott et al.39 have observed that certain cultivars exhibited strong genotypic specificity in North American germplasm lines; the Manchu and AK Harrow cultivars responded well in somatic embryogenesis, and the other responding germplasm lines were genetically related to these lines. Other studies have also indicated that the genotype affected
somatic embryo induction, proliferation and maturation14,16,40,41
. The Indian germplasm contains more than 1500 lines, and only 70 popular genotypes were selected in the present study to identify those Indian cultivars/genotypes that responded the best to treatment. The screening of Indian genotypes will be useful in the selection of cultivars for genetic transformation and subsequent breeding programmes.
Histology and photomicrography—Histological studies show that globular structures were originated from the basal cells (by sub-epidermal divisions) of the cotyledonary mesophyll tissues and the protuberance was covered by the single layer of the epidermal cells (Fig. 2c). These globular structures elongated and differentiated into somatic embryos.
The globular structures that formed somatic embryos, exhibiting clear bipolarity, were observed at the sub- marginal region of explants (Fig. 2d). Histological studies of secondary somatic embryogenesis showed that, the apical cells of the epidermal layer of the primary embryos undergone division and produced secondary somatic embryos (Fig. 2 e–i). Secondary somatic embryos formed directly from the primary embryos and they became clearly visible after 21 days (3 weeks) of culture. The anatomical analysis of cotyledonary stage somatic embryo differentiated on LHM containing NAA (10.74 µM) showed regular vascularisation along with hypocotyledonary axis and the presence of root meristems and shoot meristems (Fig. 2j)
In conclusion, an efficient somatic embryogenesis protocol was developed for Indian soybean cultivars and 71 Indian cultivars were screened for somatic embryo induction. Of the 71 cultivars tested, six cultivars showed better response to somatic embryo induction than remaining cultivars and those 6 cultivars may be used to transfer the desirable genes to improve the quality and quantity of the soybean.
Fig. 2—Histological analysis of somatic embryo development in Indian soybean cv. Pusa 16. a: Cross section of immature cotyledon explant showing epidermal layers (bar = 300 µm), b:
Immature cotyledon explant showing divisions of cells at the epidermal layer after 3 days of culture (bar = 300 µm), c: Globular structures resulted from sub-epidermal divisions after 7 days of culture (bar = 300 µm), d: Globular embryos showing continuous epidermis with explants (bar = 300 µm), e: Secondary embryo originating from apical layer of primary embryos (bar = 300 µm), f–i: Different stages of secondary somatic embryogenesis (bar = 300 µm), j: Longitudinal section of cotyledonary stage embryo showing regular vascularisation along with hypocotyledonary axis (bar = 300 µm).
Table 4Effect of genotype in somatic embryo induction in LSEIM containing FNL macro salts, MS micro salts, B5 vitamins, 29.21 mM sucrose, 45.24 µM 2,4-D, and 20 mM L-glutamine (pH
5.8)
[Values are mean ± SE from 5 independent experiments]
Genotype tested Response (%) Mean no. of globular embryos per cotyledon*
PK 472 59.42±1.2f 3.62±0.2f
Ds 97-12 67.24±1.4b 6.24±0.4b
Gujarat soybean 60.42±1.2e 5.62±0.3c
Pusa 16 74.42±1.5a 8.45±0.2a
PK 416 63.62±1.4c 5.06±0.2d
VL soya 1 62.21±1.2d 4.26±0.2e
Each cultivar comprised of 50 explants in replicates of five.
*Results were recorded after 21 days (3 weeks) of culturing.
Percentage of response = No. of immature cotyledons responded for somatic embryo induction/Total No. of immature cotyledons cultured X 100. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5% level.
Acknowledgement
The authors are thankful to the Department of Biotechnology (DBT) of Ministry of Science and Technology, New Delhi, Government of India, for the financial support (BT/PR9622/AGR/02/464/2007).
AG is thankful to University Grants Commission (UGC), New Delhi, Government of India for providing fellowship under UGC–BSR scheme. KS thanks Council of Scientific and Industrial Research (CSIR), New Delhi for the award of Senior Research Fellowship (SRF). Thanks are due to Prof. Yong Eui Choi, Division of Forest Resources, Kangwon National University, Chunchon, South Korea for help in histological sectioning.
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