Effect of Plant Growth Regulators, Modified Nitrogen and Carbohydrate Sources on Somatic Embryogenesis of Rice (Oryza sativa L.) Cultivar BPT-5204

Rice is a staple food for more than half the world’s population. To safeguard the production and to fulfill the demand of ever growing population there is a need to increase the production. The yield of rice has increased enormously due to modern technology and development of high yielding rice cultivars. But still it is 10-15 per cent lower than its potential yield due to susceptibility of high yielding rice cultivars to biotic and abotic stress. One such cultivar is BPT 5204, which is popularly known as Sambamahsuri, is a widely cultivated variety in India for its good quality and marketability. Though it has good agronomic qualities, BPT 5204 is susceptible to biotic and abiotic stresses leading to yield loss. This problem could be overcome by genetic transformation which offers an opportunity to incorporate genes of interest from a remote source into an existing high yielding cultivar (Grewal et al., 2005 and Sajid et al., 2020). The success of genetic transformation mainly depends on efficient callus induction and regeneration procedures. In rice, an efficient tissue culture system with high frequency of somatic embryo production is required for the successful regeneration and genetic transformation studies (Bijalwan and Shilpa, 2021).

The fate of call induction and somatic embryogenesis is decided by the genotype, plant growth regulators, basal salts of the medium especially nitrogen source, carbohydrate source (Tilgam et al., 2024). Among the various factors influencing somatic embryogenesis the ratio between both ionic forms (NO₃^- and NH₄⁺) plays a prominent role on the induction of somatic embryogenesis and somatic embryo growth. In addition to this, carbohydrate type and concentration have been found to play important role in different stages of the somatic embryogenesis. Specific carbohydrates may have differential effects on growth and differentiation (Verma and Dougall, 1977). Many studies reported that monosaccharides and disaccharides performed differently during the process of callus growth and somatic embryogenesis (Cunha and Fernandes-Ferreira, 1999). Therefore, the present investigation was aimed 1. To determine the best concentration and combination of plant growth regulators camma modified nitrogen sources and carbohydrate sources for excellent callus induction and somatic embryogenesis. 2. To study the morphological and biochemical changes in the callus during the process of somatic embryogenesis.

Three invitro experiments were carried out at Department of Crop Physiology, Acharya N.G Ranga Agricultural University, Agricultural college, Bapatla andhra Pradesh, India during 2019-2020 to evaluate the effect of plant growth regulators, inorganic nitrogen sources and carbohydrate sources on somatic embryogenesis of callus produced from BPT-5204. Morphological observations such as colour and appearance of was recorded in callus at 21 and 28 days after inoculation (DAI) and biochemical parameters such as ammonical nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen and protein contents were analyzed in callus at 21 DAS.
 
Explant preparation and culture conditions
 
The mature seeds of indica rice variety BPT-5204 were used as explants for callus induction. Mature dehusked seeds were surface sterilized with 70% ethanol for 4 min and rinsed with sterile distilled water for 3 times. After that seeds were further sterilized with 0.1% mercuric chloride for 7 min with intermittent shaking followed by 5-6 rinses in sterile distilled water. The sterilized mature seeds were inoculated on modified MS medium supplemented with various concentrations and combinations of plant growth regulators, inorganic nitrogen sources and carbohydrate sources. The culture plates containing explants were incubated under controlled temperature of 25±1°C in dark for 21 days. At the end of experimental period i.e., 21 days subculturing was done.
 
Experimental treatments
 
Unmodified MS medium contains 39.41 mM NO₃^- and 20.62 mM NH₄⁺ ions. In the modified medium, the nitrate form of nitrogen was supplemented in the form of KNO₃ and ammonical form of nitrogen was supplemented in the form of NH₄Cl. 


 
Biochemical parameters
 
Ammonical nitrogen content in the callus was estimated by salicylate method as given by Ali and Lovatt (1995). Nitrate content was analyzed by following the method as described by Zhao and Wang (2017). Nitrite content was determined by following the method as described by Hachiya and Okamato (2017). Nitrogen content was estimated by “Micro Kjeldhal” method as suggested by Hardarson and Danso (1993) and Protein content was determined by the method of Lowry et al., (1951).
 
Statistical analysis
 
The results obtained were statistically analyzed running the one-way analysis of variance test (ANOVA) by following the standard procedures (Gomez and Gomez, 1984). Variable number of somatic embryos per embryogenic explants was subjected to a suitable transformation to meet test assumptions.
 

Effect of plant growth regulators on somatic embryogenesis of callus
 
Morphology: Colour and appearance of callus
 
The initial step of this experiment was designed to identify the best plant growth regulator or its combination effect on somatic embryogenesis of callus. Callus initiation from cultured mature dehusked rice seed was observed within one week of the culture. In general, callus was initiated from the swollen embryonic/ scutellar region of the seed as an unorganized mass of cells, which attained maximum size within 3 weeks of dark incubation.

The effect of PGRs on morphology of the rice seed callus was recorded at 21 DAI and 28 DAI (after subculturing) are presented in the Fig 1 and 2. On plain MS medium (control) without any PGR there was no callus initiation or development, but the seeds germinated, giving rise to plumule and radical. With 2,4-D at 10 micro (MS 10D) white, compact and rhizogenic callus was observed at 21 DAI, which on sub-culture showed extensive root differentiation with less callus at 28 DAI. The appearance of callus produced on MS 10D2.5K medium was yellowish white, dry, compact at 21 DAI and the texture of the callus remained compact even after sub culturing at 28 DAI. Whitish yellow, compact calli were observed on MS 10D2.5B medium at 21 DAI and root differentiation was observed in calli after sub-culturing at 28 DAI. Results showed that when MS medium was supplemented with both 2,4-D and kinetin i.e., MS 10D2.5K, yellowish white compact callus with higher embryogenic potential was generated compared to supplementation of either 2,4-D (MS 10D) alone or 2,4-D and BAP (MS 10D2.5B). Similar findings of 2,4-D and kinetin promoting embryogenic callus was reported in rice (Wani et al., 2011). Abiri et al., (2017) reported that auxins increase callus quality whereas cytokinins like kinetin increase the callus growth rate and they also stated that 2,4-D alone was not suitable for proliferation of callus which supports the current experimental results.



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Biochemical changes in the callus
 
The endogenous levels of ammonical nitrogen content, nitrate, nitrite, total nitrogen and protein content were analyzed in callus at 21 DAI (Table 1). Ammonical nitrogen content, nitrate, total nitrogen and protein contents were lower in the callus produced on MS 10D2.5K medium. Similar findings of lower total nitrogen content in the embryogenic callus was reported in Medicago arborea L.  by Gutierrez et al., (2010). In sugarcane, Neves et al., (2003) reported low protein content in the embryogenic callus as compared to non-embryogenic callus, which was attributed to protein utilization during embryo induction.



From the results obtained from the first experiment on PGRs, MS 10D2.5K medium was selected as the control (MSP) for second experiment to study the effect of nitrogen source of the medium on somatic embryogenesis, since it resulted in compact callus with embryogenic appearance.
 
Effect of modified inorganic nitrogen sources on somatic embryogenesis of callus
 
Morphology of callus-colour and appearance
 
Effect of modified nitrogen sources on rice seed derived callus morphology at 21 DAI and at 28 DAI is presented in Fig 3 and 4. In the control medium i.e., MSP, callus was light yellow and compact at 21 DAI which remained compact even after subculturing at 28 DAI. Callus produced on MSP 60N medium was yellowish brown initially which gradually became necrotic and soft textured at 21 DAI and subculturing was not done since the callus was minimal. Light yellow, differentiating but minimal calli were observed on MSP 60A medium at 21 DAI. The morphology of calli produced on MSp 20A:20N medium was off-white, soft and friable at 21 DAI, which remained the same even after subculturing at 28 DAI. Creamish to light yellow and compact calli were formed on MSP 40A:20N medium at 21 DAI, which developed into compact embryogenic calli with globular embryos on surface after subculturing at 28 DAI.





The current results concur with the published reports of Halperin and Wetherell (1965) who observed that reduced nitrogen in the form of NH₄⁺ was capable to induce embryos while the oxidized form (NO₃^-), even at a high concentration of 60 mM was ineffective. Grimes and Hodges (1990) reported that calli became necrotic and died in the rice cv IR54 when immature embryos were grown on medium containing KNO₃ as a sole nitrogen source which support our current findings.
 
Biochemical changes in callus
 
The endogenous levels of ammonical nitrogen content, nitrate, nitrite, total nitrogen and protein content were analyzed in callus produced on different nitrogen sources at 21DAI (Table  2).



Highest ammonical nitrogen content was recorded in the callus produced on MS_P 60A medium, while lowest in callus produced on MS_P medium. Highest nitrate nitrogen content was recorded in callus of MS_P 60N at while the lower was recorded in MS_P 40A:20N which was on par with MS_P each other. Lower levels of nitrate nitrogen in embryogenic callus was reported by Gutierrez et al., (2010) in Medicago arborea L. This decrease might be due to nitrate metabolism which is important for callus differentiation as suggested by Cunha and Fernandes-Ferreira, (1999) which support the current findings.

Highest nitrite nitrogen content was recorded in the callus of MSP 60N in 21 days old callus  and this higher nitrite content possibly resulted in yellowish brown, necrotic callus on medium containing nitrate alone (MS_P 60N). Yatazawa and Furuhashi (1968) reported that rice cells were unusually sensitive to nitrite, which accumulated during growth of rice cells on medium containing nitrate which support the current findings. The total nitrogen content and protein contents were content was lowest in the 21 days old callus produced on MS_P 40A:20N medium. Similar findings of decrease in the levels of total nitrogen content in the embryogenic callus were reported in Medicago arborea L. (Gutierrez et al., 2010). Similar findings of decline in the protein levels during the formation of embryoids were reported in cumin by Dave and Batra (1995). This might be due to utilization of high protein content present initially at the site of embryoid formation and the exhaustion of this macromolecules was probably to synthesize proteases and amino acids after embryoid formation as suggested by them.

From the results obtained from the second experiment, MS_P 40A:20N medium was selected as the basis to study the effect of different carbohydrate sources in the medium on rice somatic embryogenesis because the calli produced on it were compact with distinct embryos on the surface.
 
Effect of carbohydrate sources on somatic embryogenesis  of callus
 
Morphological characters and biochemical constituents were evaluated in the callus rice seed derived callus produced on selected medium MS_PN i.e., MSPN40A:20N with different types of carbohydrate sources viz., MS_PN (Control), MS_PN + Maltose (MS_PN M), MS_PN + Glucose (MS_PNG), MS_PN+ Fructose (MS_PNF), MS_PN1:1 Glucose: Fructose (MS_PN1G:1F).
 
Colour and appearance of callus
 
Effect of different types of carbohydrate sources on morphology of callus generated from rice seeds at 21 DAI and 28 at DAI are presented in the Fig 5 and 6. Either at 21 DAI or at 28 DAI there was not much difference in the colour and appearance of the calli generated under different carbohydrate sources in the media from rice seeds. At 21 DAI except the difference in size, all the treatments resulted in calli which was yellow to off-white to white, which on subculture developed into embryogenic callus with visible globular embryos on the surface at 28 DAI.




 
Biochemical parameters in callus
 
Various nitrogenous constituents were estimated in the calli generated from dehusked rice seeds cultured on medium supplemented with different types of carbohydrate sources at 21 DAI (Table 3). Highest ammonical nitrogen content was recorded in the callus produced on MS_PN G medium while, it was lowest in the callus produced on MS_PN M at 21 DAI. There were no significant differences in the nitrate content of calli produced on MS_PN and MS_PN M, MS_PN 1G:1F and MS_PN F media at 21 DAI while, highest nitrate nitrogen content was recorded in the calli produced on MS_PN G. Similar lower levels of nitrate nitrogen in embryogenic calli were reported by Gutierrez et al., (2010) in Medicago arborea L. This decrease might be due to nitrate metabolism which is important for callus differentiation as suggested by Cunha and Fernandes-Ferreira (1999).



Highest nitrite content was recorded in the calli produced on MS_PN while, it was lowest in the calli produced on MS_PN M and MS_PN F media which were on par with each other at 21 DAI. Lowest total nitrogen content was recorded in the calli produced on MS_PN M medium while, it was highest in the calli produced on MS_PN F at 21DAI. Similar findings of decrease in total nitrogen content in the embryogenic callus was reported in Medicago arborea L. (Gutierrez et al., 2010). Lowest protein content was reported in the calli cultured on MS_PN M. The treatments viz., MS_PN G and MS_PN 1G:1F didn’t differ significantly with respect to protein content of the callus at 21 DAI while, showing significantly higher values than the calli produced on MS_PN, MS_PN F and MS_PN M media. Similar findings of decrease in the level of protein during the induction phase of somatic embryogenesis was reported in Acca sellowiana (Cangahuala-inocente et al., 2014). This reduction might be due to consumption of proteins in order activate the cellular metabolism for the establishment of embryogenic competence.
 
Number of somatic embryos/explant
 
Significant differences were observed in the rice seed derived callus with respect to number of somatic embryos per explant on the medium supplemented with different types of carbon sources at 28 DAI (Table 4). Highest numbers of somatic embryos were observed in the calli produced on MS_PN M. Lowest number of somatic embryos were reported in the callus produced on MS_PN G. Similar results showing superiority of maltose over sucrose on somatic embryo production were reported in apple by (Daigny et al., 1996). In Cymbopogon schoenanthus maltose containing medium recorded highest mean value of somatic embryos followed by sucrose containing medium which support our current findings (Salam et al., 2015).

From the above results, it is more evident that MS medium supplemented with 10 µM 2,4-D and 2.5 µM kinetin resulted in compact callus with embryogenic appearance. The presence of kinetin in the medium along with 2,4-D might have enhanced the proliferation of cells resulting in the more compact calli. The MS medium containing ammonium and nitrate nitrogen in the ratio of 2:1 at a concentration of (40 : 20) mM along with best combination of plant growth regulators (MS_P) resulted in compact callus with globular embryos on the surface. Maximum number of somatic embryos were observed with 3% maltose as carbon source in the MS media with best composition of plant growth regulators and nitrogen source (MS_PN M). This media composition can be further used for development of climate resilent varieties through genetic transformation studies.

In the present study, various biochemical events that takes place during production of somatic embryos were also clearly studied. Biochemical parameters such as ammonical, nitrate, nitrite, total nitrogen and protein contents were found to be decreased in the callus produced on MSPN M medium indicating the rapid nitrogen metabolism during the process of somatic embryogenesis. Lower protein, total nitrogen and low nitrate contents of callus can potentially be used as biochemical markers for somatic embryogenic potential in indica rice (Oryza sativa L.) which should be used along with visible confirmation of somatic embryos. This work can be extended to know the changes in the activities of enzymes of nitrogen metabolism which invariably play a major role in the structural and metabolic development of somatic embryos in rice and further into the accumulation of embryogenesis specific proteins and other macro molecules.

All authors declared that there is no conflict of interest.