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An experiment was assessed for measuring the effect of partial physical desiccation on plant regeneration efficiency in scutellum derived embryogenic callus of rice (Oryza sativa L.) variety Super basmati. A number of callusing cultures were developed, while efficient callus induction was observed on MS basal medium supplemented with 2.0 mg/L 2,4-D. The calluses were proliferated on the same medium for 3-weeks than shifted to dehydration desiccation treatment for 72 hrs. The desiccated calluses were cultured on different medium for somatic embryogenesis and plant regeneration. A medium with 2.0 mg/L NAA, 10.0 mg/L ABA, 2.0mg/L KT was considered best for somatic embryogenesis only, not for further plant development. After 10 days, regenerate-able (differentiated) calluses were sub-cultured on medium with various concentrations and types of carbohydrates (carbon source) in 1MS2j medium. A large number of plantlets 14.51±2.81 and 8.56±2.90 plants/callus were regenerated via chemical desiccation, on MS with 3% maltose + 3% sorbitol and 6% sucrose respectively, while through dehydration only on simple MS (3% sucrose), 11.23±3.22 plants/callus were developed. Through dehydration and chemical desiccation plant regeneration rate was higher than the calluses cultured on simple MS medium. It was also concluded that the plants regenerated in the presence of PGR after somatic embryogenesis, >25% plants were sterile. This protocol may enable to regenerate maximum numbers of normal and fertile plantlets of super basmati rice within 3-months.
Key words: Plant regeneration efficiency, Oryza sativa L., Super basmati, relative callus proliferation rate, somatic embryogenesis, physical desiccation.
Abbreviations: 2,4-D-2,4-dichlorophenoxy acetic acid; 2-IP-2-isopentenyl adenosine; MS-Murashinge and Skoog medium; PGR-plant growth regulator; TDZ-thidiazuron, NAA-α-Napthaleneacetic acid; KT-kinetin; IAA-Indoleacetic acid, Ci-callus induction medium, Rg-plant regeneration medium.
Rice is a staple food source for more than half of the mankind from the ancient days. It is a member of the five most important world's cereal crops. About 92% of total rice is produced and consumed in Asia (Khush, 1997). In Pakistan, during 2007 rice production was 5.49 million tones of which 2.5 million tones corresponded to basmati rice (http://www.fao.org/es/esc/en/index.html). Despite this, a number of abiotic and biotic factors have been limiting its productivity? Genetic transformation is an invaluable tool to develop natural resistance in plant against all yield limiting factors, but it depends on an efficient in-vitro plant regeneration system from a single cell.
The callus initiations in cultured parts (explant) of a species, its proliferation than subsequent regeneration are prime steps in tissue culturing among cereals (Snezana et al., 2005). Each step is to be manipulated by biotechnological means to design an efficient protocol for plant regeneration in any plant species. The potential for callus formation and its regeneration have been reported to be a varietals specific characteristic (El-Bakry and Ahmed, 2002; Barry-Etienne et al., 2002). Meanwhile, the strategies to improve plant regeneration frequency in cereals, including rice, have been steadily evolving during the last decade (Kyozuka et al., 1988; Datta et al., 1992; Raman et al., 1994; Itoh et al., 2006). Different tissues of rice plant have been used as an explant (Bhaskaran and Smith, 1988) for callus induction. However, produced callus has limited totipotency for its successful regeneration (Maggioni et al., 1989; Vyas et al., 2009), which is depending on a number of bio-physical characteristics (Droste et al., 2005). During plant regeneration from embryogenic callus; somatic embryo is an intermediate stage between un-differentiated callus (somatic cells) and seedlings. It is differentiated and meristematic form of a somatic (callus) cell developed through a series of complex morphological and cellular changes (Laux and Jurgens, 1997; Helariutta et al, 2000; Wei, 2001; George et al., 2008). Specific cellular changes can induce embryo and its maturation, which is one of the main barriers for the success of somatic embryogenesis (Walker and Parrott, 2001; Li et al., 1998; Misra et al., 1993; Tremblay and Tremblay, 1995; Bozhkov and Arnold, 1998). The factors that determine or bring bio-physical changes in the cells to accumulate, enough storage materials (Bozhkov & Arnold, 1998) and desiccation tolerance (Blackman et al., 1992) for the its conversion to embryos (Murthy et al., 1998; Fry, 1995) and than its maturation (Thomas, 1993; Merkele et al., 1995). Somatic embryogenesis can be influenced even by developing low osmotic potential in the maturation cultures (McKersie and Brown, 1996; Walker and Parrott, 2001). Commonly, carbohydrates are used in the cultures as carbon sources for the development of tissues (Iraqi and Tremblay, 2001) into plantkets. These compounds can be acting as a dual in function as somatic embryogenesis (playing a role as osmotica) and being a nutrition source (Li et al., 1998) in the cultures. In many species it is observed that an increased sugar concentration generally improves somatic embryo maturation (Tremblay and Tremblay, 1995; Li et al., 1998; Iraqi and Tremblay, 2001). Plant regeneration from embryogenic callus has achieved initially in japonica rice varieties (Nishi et al., 1973). Successful regeneration of fertile plants has been limited in Indica rice varieties (Kyozuka et al., 1988; Raman et al., 1994). It is also a reason that the progress towards the transfer of useful genes in Indica rice has been going to at very slow rate.
Of course, normal somatic embryogenesis may occur, but its maturation and than plant regeneration is dependent on specific physical stresses. Partial as well as temporal stresses may result into an improvement for minimizing the rate of abnormal plant development. Partial physical desiccation treatments have been reported to be beneficial for embryogensis as well as plant regeneration in several plant species (De Gloria et al., 2000; Mingozzi et al., 2009; Tyagi et al., 2007). The dehydration of cell suspension derived from the embryogenic calluses (Jain et al., 1996; Chand and Sahrawat, 2001; Tsukahara and Hirosawa, 1992; Zhu et al., 1996) can increase plant regeneration efficiency. On the basis of this idea, we require to observe the effect of chemical desiccation through maltose and sorbitol supplement (to increase osmotic pressure) in place of sucrose in the plant regeneration medium, with an additional dehydration desiccation treatment on proliferating calluses prior to their regeneration.
In this manuscript we had designed a number of cultures to assess the comparative effect of different hormones under partial physical desiccation (via both chemical desiccation and dehydration desiccation) stresses on the efficiency of plant regeneration from scutellum derived embryogenic callus of recalcitrant Indica rice variety "Super basmati". This work may be of invaluable in future for further improvement in rice or other cereal crop to establish their genetic transformation system.
Materials and Methods
Plant material and sterilization
Mature and healthy seeds of rice (Oryza sativa L.) variety Super basmati were selected, dehusked and surface sterilized with 50% (1:1, v/v) commercial bleach (5.25% NaOCl) by stirring on magnetic stirrer for one hour than washed (3x5 min) with sterile distilled water. Twenty to thirty surface sterilized seeds were cultured on a number of callusing medium. The cultures were incubated at 25 ± 2°C in dark.
For callusing a number of medium was maintained such as MS basal medium [MS (Murashige and Skoog, 1962) basal salts, B5 vitamins (Gamborg et al., 1968) and 3% sucrose] supplemented with 2.0 mg/L 2,4-D separately or in a combination with 500 mg/L proline and 2 mg/L NAA. Each type of medium was solidified with 1% (w/v) purified granulated agar (Difco) and its pH was adjusted between 5.7-5.8 prior to sterilization.
The callus proliferation rate (%) was measured by culturing ~50mg callus (after 7-days) from *MS2a to all other medium including itself (*MS2a- 1MS2j) for 3-weeks. It was calculated by applying formula:
Where FWi - initial fresh weight (50mg), FWf - final fresh weight of callus
Physical desiccation treatment
Partial physical desiccation was carried out by transferring embryogenic callus from *MS2a (somatic embryo induction) to sterile empty petri dishes containing two sterile whatman-1 filter papers for their dehydration desiccation. The petri-dishes were sealed with parafilm and kept at 25±2°C in dark for 72h. After desiccation treatment, calluses were transferred to different plant regeneration medium. For chemical desiccation experiments, c. sucrose (6%) and d. sorbitol (3%) + maltose (3%) were added in the plant regeneration medium (PRM) in place of 3% sucrose (w/v). The PRM (comprised on MS salts, B5 vitamins and 3% sucrose) was kept as a control PRM on which embryogenic calluses was cultured without desiccation treatment (culture). After dehydration desiccation treatment, the calluses were also cultured on PRM (b).
A number of cultures were maintained for plant regeneration. One specific culture (1MS2j) was also established by culturing callus from 1MS2h (after 10-days). Plant regeneration was observed after 30-days in each of the cultures i.e. a, b, c, d.
Root induction and plant hardening
The regenerated plantlets were transferred to culture tubes for shoot elongation and root induction containing MS basal medium. After 2 weeks, rooted plantlets were transferred to soil in earthen pots covered with polythene bags for plant hardening. The plants were finally shifted to the green house after 7-days.
All cultures were incubated at 25±2°C under hrs day and light conditions (light intensity 15 µmol m-2 s-1) provided by white fluorescent tubes (36 W/54, 6500 K) in the growth room till plants hardening on the soil medium.
Growth regulators sterilization
In all cultures, a number of plant growth regulators were used. They were sterilized differently depending upon their stability i.e. heat labile growth regulators i.e. IAA (indoleacetic acid) and ABA (abscisic acid) were filter sterilized by using sterile Millex-GS, 0.22 µm filter unit, while others, i.e. a-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) kinetin (KT) and 2-isopentenyl adenine (2-ip), thidiazuron (TDZ) were added in the medium (from stock) before autoclaving.
Statistical data analysis
The experiment was arranged as a randomized complete block with 7 replicates per treatment during callusing or plant regeneration. Data were analyzed using the SAS program Version 6.11 (SAS Institute, Cary, NC). A probability level of 5% (=0.05) was chosen for all statistical inferences.
Results and Discussion
Today cereal's yield potential has been limited due to a number of biotic and abiotic stresses. Present yield graph of cereals is not fulfilling the increasing demand of human population. Not any conventional method is available for crop improvement in very short time. While modern biotechnology could enable the cereals to resist against specific environmental stress and to express its potential in the form of yield. Among the modern techniques, genetic engineering is totally dependent on efficient plant regeneration system of a crop. The initial step is callusing from any tissue (explant) of plant, which is an important step to establish its tissue culture system. Similarly in case of rice has been considered as a most critical step. A number of medium has been tried for callus induction and its proliferation, such as MS supplemented with 2,4-D (2.0 mg/L), NAA (2.0 mg/L) and proline (500mg/L) separately or in combination were maintained (*MS2). Within 7-days, callus become visible (induced) from the scutellar region of seeds on each medium. Maximum callus induction frequency (92.0%) was observed on *MS2a medium. Callus proliferation rate (%) was also measured by subculturing embryogenic callus from *MS2a medium to fresh callus induction (Ci) medium as well as plant regeneration (Rg) medium. After 3-weeks, maximum 60.25% callus proliferation was observed on *MS2a. So the culture with 2,4-D only could induce efficient callus and than its proliferation (Fig 1a) in super basmati rice (Katiyar et al., 1999; Zhenyu et al., 1999; Gairi and Rashid 2004). However, use of casein hydrolysate was found to be beneficial for generation of embryogenic callus in both Japonica (Hiei et al., 1994; Khana and Raina, 1997; Toki, 1997) as well as in Indica rice varieties (Zhang et al., 1996). Similarly, the use of proline was also effective for the initiation and maintenance of embryogenic callus (Datta et al., 1992; Kishor et al., 1999).
Partial physical desiccation has been found to be promotive agent for plant regeneration (Jian, 1997; Diah and Bhalla, 2000; Chand and Sahrawat, 2001; Saharan et al., 2004). First of all the plant regeneration in embryogenic callus was started on 1MS2j culture (Fig 1) within 2-weeks, when they were cultured from 1MS2h (somatic embryo induction) medium (Fig 1b) on 1MS2j (chemically desiccated plant regeneration medium; d), while after 2-4 weeks, the plantlets were regenerated on medium on which the cultured embryogenic calluses were treated as the dehydrated desiccation (b), however after 4-weeks on the medium supplemented with 6% (c) and 3% (a) sucrose. Overall, except 1MS2jh and 1MS2i medium, the plantlets were regenerated after 7 weeks.
Within 4-weeks, the somatic embryogenic calluses on 1MS2j were entirely covered with green shoot buds. A vigorous elongation with efficient shoot multiplication was observed. Shoot multiplication rate was comparatively low in the cultures with sucrose and dehydrated desiccation treatment on 1MS2j than with maltose and sorbitol. Similarly, the physical appearance of the regenerated plantlets was comparatively not as green and healthy as the plantlets regenerated from chemical desiccation medium (1MS2j with maltose and sorbitol). The number of regenerated plantlets from a single embryogenic callus were 10.21±4.88 and 14.51±2.81 (maltose and sorbitol), but 6.18±2.11 and 11.23±1.22 (dehydrated desiccation) and 4.91±2.50 and 8.56±2.90 (6% sucrose) on 1MS2d and 1MS2j medium respectively, all these culture were considered better than cultures maintained on 3% sucrose medium. The possibility for the regeneration of such a huge numbers of plantlets was culturing by embryogenic callus on 1MS2h medium for 10 days prior to its sub-culturing on 1MS2j (Fig 1d) medium. All of these regenerated plantlets from 1MS2f (maltose and sucrose) were also observed to be fully fertile. Few plantlets were also regenerated on 1MS2f and 1MS2e (supplemented with TDZ and IAA respectively), while they seems to be not healthy as well as fertile.
Overall, the partial physical desiccation enhance plant regeneration efficiency for super basmati rice variety but the chemical desiccation instead of sucrose in MS cultures including somatic embryogenesis proved to be helpful for the improvement of both the maturation of somatic embryos and their regeneration into plantlets. It is also noted that before the plant regeneration without somatic embryo induction (1MS2h with 10mg/L ABA, NAA, KT, maltose and sorbitol) in callus is impossible (Fig 1). During somatic embryogenesis in the cultured, its growth inhibition was observed, which is due to ABA (a growth inhibitor).
The results of this study have showed that in Indica rice variety Super basmati the highest number of plantlets (14.51±2.81) were regenerated through chemical desiccation (maltose and sorbitol) treatment in comparison to the plants regenerated after dehydration desiccation or chemical desiccation (6% sucrose) treatment. The desiccation treatments induction, during or before plant regeneration were observed to be better than callus cultures without desiccation. Finally, through the applications of desiccation, all the regenerated plantlets were fertile.
The recalcitrance of Indica rice varieties to tissue culture has been a major stumbling block for their transgenic development. In addition, to the fact that currently agronomic Indica rice improvement totally depends only on the Japonica rice varieties, which could potentially lead to a genetic bottleneck problem. This tissue culture protocol for Super basmati rice, we have developed/produced a high percentage of regenerable somatic embryogenic calluses, in the presence of a combination of different hormones in the somatic embryogenesis medium and through partial physical desiccation (in the absence of PGR), a large number of plantlets were regenerated. Both 1MS2h and 1MS2j media, in particular, produced excellent results, both for the development of somatic embryos (PGR) and for efficient plant regeneration (partial physical desiccation). However, when plant regeneration was carried through partial physical desiccation in the presence of PGR, >25% of total regenerated plantlets were sterile.
We are currently testing the embryogenic potential for plant regeneration efficiency by using the protocol described in this paper for the purpose of establishing its genetic transformation system. Super basmati rice is an agronomically improved cultivar with good yield and highly palatable, so it will have little genetic drift in transgenic back cross programs as compared to other Japonica rice varieties. Therefore, regeneration of plants through somatic embryogenesis in Super basmati rice constitutes a significant step towards broadening the genetic base of transgenic rice cultivars.
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