1. INTRODUCTION
Rice is the major staple food of more than half of the world population. Feeding hungry with nutritious rice seems to be a lasting solution to food and nutritional security. Rice is grown in more than 120 countries, with a total harvested area of approximately 167 million hectares with a production of 496.1 mill tons in 2019–20 (https://www.statista.com/statistics/271972/world-husked-rice-production-volume-since-2008/). China and India are the top leading countries which contribute 50% of global rice production. Asia alone meets 90% of global milled rice requirement. At least 60% increase in food production is needed in next 30 years to feed the world (https://www.eitfood.eu/blog/post/sustainably-feeding-the-world-in-2050-are-efficiency-and equity-the-answer) which is indeed a challenging task. Zn is a trace mineral and it serves as cofactor of more than 300 enzymes involved in cellular metabolism [1]. In animals, Zn deficiency leads to loss of immunity to diseases, stunted growth, impaired learning ability, wound healing, and reproduction; and increased risk of infection, DNA damage, and cancer [2]. Therefore, there is a need for Zn-biofortified rice in the food chain. In plants, Zn is needed for plant growth and resistance to biotic and abiotic stresses [1]. Grain Zn content is a complex polygenic trait with high G x E interaction [3]. Available Zn status in soil, influx to roots, presence of Zn-transporter genes, exudation of phytosiderophores, inherent physiological mechanism of Zn uptake, transport, and remobilization to sink (seed), and metal homeostasis determine the grain Zn content. Innovative breeding strategy coupled with biotechnological approaches can pave the way for development of high Zinc rice variety.
In cereals, various in vitro culture techniques are being applied for varietal development among which matured dehusked seed culture is often used for genetic transformation and creation of novel genetic variants. However, its application is limited by genotype, media supplements, and culture conditions [4] to sustain growth of calli, subsequent plant regeneration and survival as fertile plants. It is often difficult to induce embryogenic calli and to regenerate plants from the callus cultures specially those belonging to Indica subspecies [4]. The recalcitrant nature of this sub-species has, in fact, been a major limiting factor in transfer of valuable genes [5] and creation of somaclonal variation and mutagen induced genetic variation(in vitro mutagenesis). An efficient callus induction and reproducible rapid regeneration system can achieve the success. Therefore, the present experiment was undertaken to optimize media supplementation and culture conditions in a popular zinc rich rice variety “Chittimuthyalu.”
Chittimuthyalu is a semi-dwarf, short bold grain land race of Andhra Pradesh (India) maturing within 135 days in wet season. “Chittimuthyalu” retains 23.45 ppm zinc, 4.05 ppm iron, and 9.31% protein in polished rice, and it is considered as quality check entry in biofortification trials of All India Co-ordinated Research Project on Rice. In addition, it has high head rice recovery (66.1%), relatively low glycemic index (~50) and suitable status of amylose content (23.81%) that fetch consumer’s preference [Supple. Table 1].
2. MATERIALS AND METHODS
2.1. Plant Material
Genetically, pure seeds of a zinc rich rice variety “Chittimuthyalu” were used for in vitro culture in this study.
2.2. In vitro Culture
Mature healthy dehulled kernels of cv. “Chittimuthyalu” was washed with 2% bavistin (w/v) for 30 min and surface sterilized with 70% ethanol for 2 min followed by washing (2×) with sterilized distilled water with a drop of Tween 20 with continuous shaking for 10 min. Further, the seeds were treated with 0.1% (w/v) HgCl2 solution for 6 min followed by rinsing (5×) with sterile distilled water and blot dried on sterilized filter paper before inoculation on culture medium. Sterilized kernels were aseptically cultured in modified MS medium with 2.5 mg/l 2, 4-D + 0.5 mg/l Kn for callus induction. Calli induced were transferred to modified MS (R)medium supplemented with 0.5 mg/l NAA + 2.0 mg/l BAP to study plant regeneration response. The pH was adjusted to 5.7 with 0.1N NaOH or 0.1N HCl after addition of the plant growth hormones and autoclaved at 121°C for 15 min. All the cultures were maintained in a sterilized culture room and incubated at 25±1°C and relative humidity of 60±5% under specified photoperiod conditions for standardization.
Sub-culturing of calli into fresh MS media (MS with 2.5 mg/l 2, 4-D + 0.5 mg/l Kn for organogenic calli and MS with 2 mg/l 2, 4-D + 0.5 mg/l Kn for somatic embryogenic calli) was done at an interval of 4 weeks for maintenance of callus growth. For this, the calli were partially desiccated in two layers of whatman-1 filter paper on Petri dish sealed with parafilm and kept at 25±1°C in dark for 48 h to attain partial dehydration. Regenerated plantlets were transferred to half-strength basal R medium supplemented with 1 mg/l NAA and varying levels of BAP (0.1–0.5 mg/l) for rhizogenesis. Each step of in vitro culture was repeated, at least twice. Each experiment comprising individual culture variable at varying levels was laid out in completely randomized design with 24 replicates. Observations were recorded for callusing response, callus growth, morphogenetic potential, and plant establishment; and analyzed statistically as per Dafaallah [6].
2.3. Culture Variables
The culture variables included key media supplements, for example, casein hydrolysate (CH) (0–1000 mg/l), proline(25–500 mg/l), carbon sources (glucose, sucrose and maltose: 2–4% each), gelling agents (agar, gelrite, phytagel, and their combinations), and culture conditions, for exa,ple, photoperiods (light/dark : 0/24–24/0 with increment of 4 h exposure to light), desiccation (no and partial desiccation), and age of calli (after 1–4 passages of subculture) for optimization of in vitro culture of cv. “Chittimuthyalu.”
3. RESULTS AND DISCUSSION
Zn content is a highly complex trait, and the variety “Chittimuthyalu” is known to have stable performance for grain Zn content over years; hence, the genotype seems to be a best candidate material to explore genetic variation at cellular level (somaclonal variation) or genetic manipulation using in vitro mutagenesis and genetic transformation for biotic and abiotic stresses and yield per se. For this, optimization of culture variables is a priori to develop a high throughput rapid plant regeneration system.
Callus induced from mature seed scutellum can follow either organogenic or somatic embryogenic plant regeneration on a suitable modified MS media. In this study, a large number of small embryoids were formed on each callus. Many of them did not develop and as such their germination was poor and irregular. Nevertheless, some callus gave rise to more than 10 plantlets. Hence, optimization of culture variables was needed to improve the number of viable plants from embryogenic calli. In fact, we tested a series of culture variables at varying levels as step by step independent experiments to optimize callus induction and morphogenetic potential of an upland rice variety “‘Chittimuthyalu” in MS media with 1000 mg/l NH4NO3 + 2830 mg/l KNO3 as nitrogen source. There was a continuous improvement in callusing and plant regeneration response in subsequent follow-up steps. The optimized level of each culture variable is shown in Table 1. Figures 1 and 2 depict high throughput callusing response, growth of callus and follow-up green plant regeneration under optimized combination of media supplementation and culture conditions.
 | Table 1: Optimum MS media composition and culture conditions for embryo culture in rice cv. “Chittimutyalu”. [Click here to view] |
 | Figure 1: Organogenic callus induction (a and b) and callus proliferation upon subculture(c) in modified MS + 2.5 mg/l 2, 4-D + 0.5 mg/l Kn with addition of 500 mg/l CH, 150 m/l proline, 3% sucrose and 0.3% agar + 0.2% phytagel under complete dark condition in rice cv. “Chittimutyalu.” Somatic embryogenic callus induction (d and e) in modified MS + 2.5 mg/l 2, 4-D + 0.5 mg/l Kn and callus proliferation upon sub-culture (f) in modified MS + 2 mg/l 2,4-D+ 0.5 mg/l Kn with addition of 500 mg/l CH, 150 m/l proline, 3% sucrose and 0.3% agar + 0.2% phytagel under complete dark condition in rice cv. “Chittimutyalu.” [Click here to view] |
 | Figure 2: Organogenic (a and b) and somatic embryogenic (nodular and friable) (c and d) shoot bud differentiation and plantlet regeneration in modified MS + 0.5 mg/l NAA + 2 mg/l BAP with addition of 500 mg/l CH, 150 m/l proline, 2.5% sucrose and 0.3% agar + 0.2% phytagel under 12 h photoperiod using 4 week old partially desiccated calli in rice cv. “Chittimutyalu.” [Click here to view] |
3.1. Optimization of Media Supplementation
3.1.1. CH
CH determines the quality and quantity of callus proliferation [7]. CH induces somatic embryos formation in callus culture of indica rice [8]. In the present investigation, CH concentration till 200 mg/l did not result any difference than media with no CH. However, CH at 500 mg/l elicited satisfactory callus induction (79.9±0.42%) and plant regeneration through organogenesis (62.8±0.55%) and somatic embryogenesis (74.4±0.35%) [Supple. Table 2]. At such concentration, calli were induced as early as 10th day of primary culture [Table 1] and were proliferated with rapid growth. However, Abiri et al. [9] reported that a much lower concentration of CH (100 mg/l) stimulated somatic embryogenesis and plant regeneration in Malaysian rice cv. MR 219.
3.1.2. Proline
Auxin-induced somatic embryogenesis in the presence of proline is well documented [8]. Proline is reported to have role in the initiation and maintenance of embryogenic calli [10]. Free proline acts as an osmoticum, a nitrogen storage pool and source of NADP + necessary for rapidly growing embryos. The tissues grown in controlled condition in artificial nutrient media undergo a kind of in vitro stress simulating to drought and/or cold stress. The growth of calli will naturally be hampered and in some recalcitrant species, callus induction, its growth, and nature of calli become extremely affected. Proline accumulation is a common phenomenon in response to abiotic stresses. Proline acts as osmotic stabilizer. Plant species sensitive to abiotic stresses, accumulate lower level of proline under stressful condition and these species need extraneous supplementation of proline to the medium to sustain normal growth and development of calli.
In the present investigation, addition of proline with increased concentration elicited marginal increase in callus induction frequency and it was highest (82.5±0.15%) at 150 mg/l with optimum growth of calli [Table 1]. Organogenic response remained more or less unaltered (66.5–66.8%) with increased concentration up to 100 mg/l and it was suddenly increased to 68.8 + 0.63% at 150 mg/l, but it marginally improved somatic embryogenic response (76.8 + 0.65%) [Supple. Table 3]. Somatic embryogenesis and regeneration was reported to be enhanced when proline was added to the medium along with 2,4-D [11]. Saharan et al. [12] and Pawar et al. [13] successfully induced somatic embryogenic calli in MS basal medium containing elevated level of proline (500 mg/l) and 2.0–2.5 mg/l 2, 4-D. However, Abiri et al. [9] reported much lower concentration of proline (50mg/l) to stimulate somatic embryogenesis and plant regeneration in Malaysian rice cv. MR 219.
3.1.3. Carbon source
Sucrose – a disaccharide of glucose and fructose serves as the chief source of carbon and energy. Besides, it has role in cellular osmotic adjustment by altering cell wall properties [14] and modulation of gene expression by acting as chemical signal in plants [15]. It remains metabolically stable at pH 5.5–5.8 and even at autoclave conditions while sterilization of the media. It is accumulated in the cell as starch which gets converted to simple sugars by sucrolytic enzymes and acid invertase in the cell to meet heavy demand of energy during callus growth and morphogenetic differentiation [16].
In the present study, glucose, sucrose, and maltose at varying concentrations (2.0–4.0%) were tried [Supple. Table 4]. Glucose at 2.5% induced callus as early as 8 days of primary culture, but sub-culturing was needed at short intervals (12–15 days) to maintain growth of calli. In this context, organogenesis induced favorably by 3% sucrose whereas, still lower concentration (2.5%) of it proved to be better for somatic embryogenic plant regeneration [Table 1]. However, increased sucrose content resulted decline in callus induction frequency and morphogenetic response possibly due to decrease in the cellular water content. Thus, sucrose seems to be the best source of carbon for plant regeneration, followed by glucose and maltose [17] and it is an absolute requirement for embryogenic callus formation [18] in Japonica rice.
3.1.4. Gelling agents
A solidifying agent is universally added to the medium to support (or hold) the explants and calli at a stationary state on the medium. Agar is widely used for in vitro culture of mature seeds in rice and other crops though other gelling agents, for example, gelrite and phytagel are used either singly or both or in combination with agar in certain cases to standardize the medium [19,20].
In the present investigation, 0.3% agar + 0.2% phytagel revealed rapid callus growth and highest callusing response (85.2 + 0.51%) [Table 1]. Using such a combination and concentration of gelling agents, organogenic and somatic embryogenic regeneration response (78.2 ± 0.45% and 82.0 ± 1.02%, respectively) was also appreciably increased over 6% agar used alone [Supple. Table 5]. This is in agreement with Sahoo et al. [19].
3.2. Optimization of Culture Conditions
3.2.1 Photoperiod
In general, callus cultures from caryopsis of rice are incubated under dark until onset of shoot morphogenesis [20,21]. However, Luo et al. [22] observed slightly better calli that grew well in the light than in the dark condition. Revathi and Pillai [23] and Roy et al. [24] observed satisfactory callus induction and plant regeneration in dark but, Wani et al. [25] obtained similar result under continuous fluorescent light in growth camber at an ambient temperature of 25 ± 2°C. In the present investigation, light intensity of 2500 lux was maintained for different photoperiod treatments at 25 ± 1°C and 68% RH. There was a progressive increase in callus induction frequency and callus growth with reduction of 4 h photoperiod per day. Complete dark was shown to be conducive for higher frequency of callus induction (89.6 ± 0.91%) as well as callus growth in the callus induction medium [Table 1]. However, 12 h photo period was optimum for organogenic response (80.2 ± 0.88%) and for maturation of somatic embryos and their follow-up plant regeneration in the regeneration medium [Supple. Table 6]. In contrast, Verma et al. [26] and Vikrant et al. [27] reported 16 h photoperiod at 25°C to be optimum for both callus induction and plant regeneration.
3.2.2. Extent of desiccation
Desiccation due to partial dehydration of regenerative calli for 48 h was found to yield positive response on both organogenic and somatic embryogenic regeneration. Comparatively, somatic embryogenic regeneration frequency increased over the organogenic response [Table 1, Supple. Table 7]. Partial air desiccation pre-treatment of calli for 45 h gave maximum green plant regeneration (76.19%) in cv. BRRI Dhan 32 and it was 2-3 fold increase than the control [28]. Similarly, Saharan et al. [12] recorded maximum shoot regeneration frequency (63%) in partially desiccated calli and it significant differed from non-desiccated calli. Further, transgenic shoots in vitro culture regenerated much faster on desiccation of calli and as such improved transformation efficiency by 77% [29]. Desiccation can also induce plant regeneration even in non-regenerative calli which might be due to elicitation of genes related to morphogenetic potential of plants. Desiccation resulting 20% loss of fresh weight of callus was reported to increase the regeneration frequency significantly in four Australian rice varieties [30]. Similar simple dehydration treatment was reported to promote somatic embryogenic plant regeneration in indica [31] and japonica [32] rice. Besides, dehydration coupled with starvation (without medium) and higher level of ABA biosynthesis might have provoked the cellular biochemical and physiological change, which is necessary for efficient plant regeneration.
3.2.3. Age of calli
Repeated sub-culturing at high concentration of 2,4-D (2.0–2.5 mg/l) in the sub-culture medium for callus proliferation may lead to increased chromosomal instability which otherwise hinders plant regeneration and plant survival. Therefore, information relating to extent of regenerability of callus cultures at different ages is a priori for recovery of higher frequency of plantlet regeneration. Sustenance of regeneration capacity until 9–10 weeks is essential to recover plants from transformed sectors after allowing two or three cycles of selection [7]. In the present investigation, about 85% of the calli showed organogenic plant regeneration and more than 88% of calli induced somatic embryogenic plant regeneration after 4 weeks of culture [Table 1]. Such calli produced profuse microtillers [Figure 3a] in regeneration medium (MS + 2 mg/l BAP + 0.5 mg/l NAA) added with 500mg/l adenine sulfate and traces of thidiazuron (TDZ) (0.01 mg/l), but microtillering capacity decreased with the age of the calli beyond 4 weeks of culture [Supple. Table 8]. In fact, the calli sustained regeneration capacity even after 16 weeks (four passages of subculture, each with 4 week duration) though there was a slow and gradual decline in regenerability and survival of plants. This may be due to in vitro induced genome stress leading to transposable element-mediated chromosomal repatterning and altered gene regulation. Further, it envisaged that the occurrence of somaclonal variation is more likely among the regenerants from long term callus cultures than direct regeneration or early generation calli.
3.3. Optimization of Hormonal Concentrations for Rhizogenesis
In contrast to somatic embryogenic plantlets, organogenic calli-derived plants usually devoided of roots and hence, required an additional step to induce rooting. Auxin alone or with very low concentrations of cytokinin is important for induction of root primordia [33]. However, it is not always true. Excised shoots when transferred to hormone-free MS [31] or half-strength MS basal medium either liquid [26] or in solid form [25] induced rooting.
In the present investigation, full strength MS basal medium failed to develop roots [Table 2]. However, half – strength MS medium was shown to initiate healthy rooting with few laterals, although % – response of rooting from the excised shoots was poor (45.08 + 0.72%). This is because low salt levels and more specifically a lower nitrogen level is usually favorable for root initiation. Therefore, an attempt was taken to optimize the hormonal combination at varying concentrations in half strength MS basal medium. NAA at 1.0mg/l with increased BAP up to 0.2 mg/l gave highest rhizogenetic response (86.6 + 0.85 %) and the excised shoots developed profuse normal rooting within a week. Further, increase in BAP (0.3 mg/l) at 1.0 mg/l NAA had shown delayed rooting with short fibrous roots, and even no rooting response at concentrations beyond 0.3 mg/l BAP. In contrast, Bano et al. [11] reported that 0.5 mg/l BAP with 0.3 mg/l IAA was sufficient for induction of roots in the regenerated plantlets.
 | Figure 3: In vitro micro tiller formation (a) using MS + 2 mg/l BAP + 0.5 mg/l NAA + 500 mg/l adenine sulfate + 0.01 mg/l thidiazuron in cv. “Chittimutyalu.” Plantlet establishment in pot mixture (b) of cv. “Chittimutyalu.” [Click here to view] |
 | Table 2: Effect of different hormonal concentrations on rhizogenesis of plantlets of cv. “Chittimutyalu”. [Click here to view] |
The plantlets with healthy roots were transferred to pot mixture (peat moss: perlite 2:1), and successfully acclimatized in glasshouse under partial shade [Figure 3b]. The plants regenerated from first few callus cultures were phenotypically normal and fertile. The in vitro protocol formulated in this study may be suitably used for in vitro mutagenesis and Agrobacterium mediated genetic transformation in zinc rich rice.
4. CONCLUSION
High throughput somatic embryogenic callus induction, proliferation, and follow-up rapid plant regeneration are in fact needed ready-in-hand for success in genetic transformation. A number of media supplements including CH, proline, various sources of carbon and gelling agents; and culture conditions, for example, photoperiod, desiccation pre-treatment, and age of calli have been verified for maximum callusing response and morphogenetic potential in a zinc rich rice cv. “Chittimuthyalu.” Besides, the role of TDZ over traditionally used cytokinins, for example, BAP and Kinetin for huge number (microtillering) of plant regeneration has been demonstrated. Supplementation of 500 mg/l CH, 150 mg/l proline, 3% sucrose, 0.3% agar + 0.2% phytagel revealed rapid callus induction and highest callusing response under dark condition. Somatic embryogenic plant regeneration was improved at 2.5% sucrose under 12-h photoperiod using 4 weeks old partially desiccated calli. The high throughput rapid somatic embryogenic regeneration system developed in this study can be amenable for genetic transformation for biotic and abiotic stress tolerance and improvement in quality traits in zinc rich rice.
5. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.
6. FUNDING
There is no funding to report.
7. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
8. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
9. PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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SUPPLEMENTARY TABLES
 | Supple. Table 1: Characteristic features of zinc rich rice cv. “Chittimatyalu” pooled over 2 years in advance varietal trial ‑2 under AICRP on rice, India in wet season. [Click here to view] |
 | Supple. Table 2: Effect of different concentrations of casein hydrolysate on callus induction and plantlet regeneration of cv. “Chittimatyalu”. [Click here to view] |
 | Supple. Table 3: Effect of different concentrations of proline on callus induction and regeneration frequencies of cv. “Chittimutyalu”. [Click here to view] |
 | Supple. Table 4: Effect of different levels of carbon source (glucose, sucrose and maltose) on callus induction and regeneration frequencies of cv. “Chittimutyalu”. [Click here to view] |
 | Supple. Table 5: Effect of different concentrations of gelling agents (Agar, gelrite, and phytagel) on callus induction and plantlet regeneration of cv. “Chittimutyalu”. [Click here to view] |
 | Supple. Table 6: Effect of photoperiod on callus induction and plantlet regeneration of cv. “Chittimutyalu ”. [Click here to view] |
 | Supple. Table 7: Effect of partial desiccation (48 h) on organogenic and somatic embryogenic plant regeneration in cv. “Chittimutyalu”. [Click here to view] |
 | Supple. Table 8: Organogenic and somatic embryogenic regeneration frequency of calli at different ages in cv. “Chittimutyalu”. [Click here to view] |