Introduction

Rice (Oryza sativa L.) is one of the major carbohydrate resources in many countries of the world, especially Asian countries, feeding more than 3 billion people and providing 50–80% of daily calorie intake (Khush 2005). Flowering, or heading date, of rice in paddy fields is well established, not only in terms of molecular biology (Hirose et al. 2006; Ishimaru et al. 2007; Chen and Wang 2008; Tsuji et al. 2008, 2011), but also as a phenotypic phenomenon (Tang et al. 2009). Rice cultivars have been classified into groups, including short-day photoperiod sensitive (SD) and short-day photoperiod insensitive. In SD rice, heading date 3a (Hd3a) protein is expressed during regulation of the SD-photoperiod-dependent flowering pathway, basically determined by expression of two essential flowering promotion genes, Hd3a and RICE FLOWERING LOCUS T 1 (RFT1), in the leaf blades and the shoot apical meristem (Abe et al. 2005; Tsuji et al. 2008). Also, a rice florigen or flowering signal has been identified, and includes the Hd3a protein, which is orthologous with the FLOWERING TIME (FT) gene product in Arabidopsis (Kojima et al. 2002). The product of the RFT1 gene, located on chromosome 6 (Izawa et al. 2003), and Hd3/FT protein signaling acts in the shoot apical meristem (Izawa et al. 2002). In the paddy field, developmental stages of rice cultivars have been well established, including seedling, vegetative, and reproductive development prior to grain harvesting (Counce et al. 2000).

In vitro flowering is a topic of interest that has already been investigated in monocotyledonous plants such as orchids (Hee et al. 2007; Sim et al. 2007; Tee et al. 2008), calla lily (Naor et al. 2004), date palm (Masmoudi-Allouche et al. 2010), bamboo and ginseng (Lin et al. 2003, 2005), Kniphofia leucocephala (Taylor et al. 2007), and Spathiphyllum (Dewir et al. 2007). From previous publications, many internal and external factors which control flowering in vitro are evident, for example, the origin of explant material, plant growth regulators (auxins, cytokinins, gibberellins, and plant growth retardants), carbohydrate to nitrogen ratio (C/N ratio), and the in vitro environment (Ziv and Naor 2006). In vitro flowering is a powerful tool used in rice breeding programs, along with in vitro fertilization and in vitro breeding (Uchiumi et al. 2007; Jung and Müller 2009). In the case of rice, there have to date been no reports on in vitro flowering. In Thailand, Pathumthani 1 (PT1: SD photoperiod insensitive) is the most popular rice cultivar grown in the irrigated zone, whereas jasmine rice, or KDML 105, is a SD-photoperiod-sensitive (providing one cultivation per annual cycle) premium quality grain, which is well-known worldwide for cooking quality, long grain morphology, aroma, flavor, and soft texture (Ariyaphanphitak et al. 2005; Laohakunjit and Kerdchoechuen 2007). The aim of this study was to develop an efficient protocol for induction of in vitro flowering in the rice crop.

Materials and Methods

Plant materials and treatments.

Seeds of Pathumthani 1 rice (O. sativa L. spp. indica cv. PT1) and KDML105 (O. sativa L. spp. indica cv. KDML105) were provided by the Pathumthani Rice Research Center (Rice Research Institute, Department of Agriculture, Ministry of Agriculture and Cooperative, Pathumthani, Thailand) and were manually de-husked, surface-disinfected once in 5% (v/v) Clorox® for 60 min, once in 30% Clorox® for 30 min, and then rinsed three times with sterile distilled water. Surface-disinfected seeds were germinated on 0.25% Phytagel®-solidified MS medium (Murashige and Skoog 1962) in 250-mL glass vessels, and then sub-cultured at monthly intervals. Alternatively, PT1 seeds were germinated (7–9 d after sowing) and then transferred to pots (15 cm diameter × 30 cm height) containing clay soil (EC = 2.687 dS m−1; pH = 5.5; organic matter = 10.4%; total nitrogen = 0.17%; total phosphorus = 0.07%; total potassium = 1.19%) for 85 d (Fig. 1A ). Experiments were conducted at the Thailand Science Park, Pathumthani, Thailand (latitude 14°01′12″ N; longitude 100°31′12″ E) between August and October 2009. In the booting stage [85 d after sowing], nodal explants (2 ± 0.2 cm lengths) were collected, surface-disinfected once in 70% (v/v) ethanol for 2 min, once in 10% Clorox® for 10 min, and then rinsed three times with sterile distilled water. Surface-disinfected nodal segments were placed on MS medium (Fig. 1B ). Two explant sources, seedling and node cuttings, were used as initial material for this experiment (Fig. 1C ). The seedling and node cutting explants were cultured on modified MS medium containing 6.81 μM paclobutrazol, 4% (w/v) sucrose, and 1.5% (w/v) agar. The culture medium was adjusted to pH 5.7 before autoclaving. The plantlets were cultured in vitro under 25 ± 2°C ambient temperature, 60 ± 5% relative humidity and 60 ± 5 μmol m−2 s−1 photosynthetic photon flux density, provided by fluorescent lamps with a 16 h d−1 photoperiod for 90 d (Fig. 1D ). Data on photosynthetic pigments, net photosynthetic rate, sugar content, in vitro flowering, and growth characters were collected. The experiment was arranged as a completely randomized design with 20 replicates (n = 20). Analysis of variance (ANOVA) was analyzed by SPSS software (SPSS for Windows, SPSS Inc., Chicago, Illinois, USA).

Figure 1.
figure 1

Protocol of in vitro flower initiation in PT1 rice (O. sativa L. spp. indica cv. Pathumthani 1); primary nodal explants (A), contamination checking (B), plantlet culture system (C), and flower developmental stage (D) after culture in modified MS medium for 90 d.

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Data collection.

Chlorophyll a (Chla), chlorophyll b (Chlb), and total chlorophyll (TC) were analyzed following the methods of Shabala et al. (1998), and total carotenoids (C x+c ) concentration was assayed according to Lichtenthaler (1987). One hundred milligrams of leaf material was collected and placed in 25-ml glass vials, along with 10 ml 95.5% acetone, and blended using a homogenizer. The glass vials were sealed with parafilm to prevent evaporation, and stored at 4°C for 48 h. Chla and Chlb concentrations were measured using a UV–visible spectrophotometer at 662 and 644 nm wavelengths. C x+c concentration was also measured by spectrophotometer at 470 nm. A solution of 95.5% (v/v) acetone was used as a blank comparator.

Net photosynthetic rate (P n) was calculated by comparing the different concentrations of CO2 inside (C in) and outside (C out) the glass vessel containing the rice plantlets. The CO2 concentrations at steady state were measured by gas chromatography (Model GC-17A, Shimadzu Co. Ltd., Kyoto, Japan). The P n of in vitro cultivated seedlings was calculated according to the method of Fujiwara et al. (1987).

Soluble sugars (sucrose, glucose, and fructose) in the leaf tissues were analyzed according to a modified Karkacier method (Karkacier et al. 2003). One hundred milligrams fresh weight tissue was ground with liquid nitrogen in a pre-cooled mortar, extracted with 1 ml nanopure water, vigorously shaken for 15 s, sonicated for 15 min, and then centrifuged at 12,000 rpm for 15 min. The supernatant was filtered through a 0.45-μm membrane and stored at −20°C prior to the measurement of the sugar content (sucrose, glucose, and fructose) using high-performance liquid chromatography (HPLC). A total of 50 μl of crude extracts were automatically injected into the HPLC system with a Waters 600 pump. On-line detection was performed using a Waters 410 differential refractometer detector, and data was analyzed with Empower software. The analytical column was a MetaCarb 87C equipped with a guard column. De-ionized water was used as the mobile phase with a 0.8-ml min−1 flow rate. Purified glucose, fructose, and sucrose were used as standards.

Flowering percentage, number of flowers per cluster, plant height (PH), number of leaves per plant (LN), leaf area (LA), fresh weight (FW), and dry weight (DW) of plantlets were recorded. Rice plantlets were dried at 80°C in a hot-air oven for 2 d, and then incubated in desiccators before the measurement of DW. LA was measured using a leaf area meter DT-scan.

Results

In this investigation, rice inflorescences successfully emerged on modified MS medium containing 6.81 μM paclobutrazol, 4% (w/v) sucrose, and 1.5% (w/v) agar, using nodal explants in the booting stage. A 60% level of in vitro flowering (inflorescence emergence) was observed for plantlets derived from nodal explants of rice (Table 1), and one florescence per cluster was demonstrated (Figs. 1D and 2B ), while flowering was absent from plantlets derived from seedling explants (Fig. 2A , Table 1). Plant height, number of leaves per plant, leaf area, fresh weight, and dry weight of in vitro plantlets derived from nodal explants decreased significantly (p ≤ 0.05), and were lower than in plantlets derived from seedlings by 8.57%, 24.24%, 23.06%, 10.54%, and 12.90%, respectively (Table 1). The growth performance of plantlets derived from seedlings was superior to that of plantlets derived from nodal explants. Moreover, the leaf color of the plantlets derived from seedling explants was a darker green (Fig. 2A ) than that of the plantlets derived from nodal explants (Fig. 2B ). Chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll, and total carotenoids (C x+c ) content in the leaf tissues of plantlets derived from nodal explants were significantly reduced, by 17.18%, 47.64%, 31.58%, and 19.87%, respectively, when compared to plantlets derived from seedlings (Table 2). The photosynthetic pigments in the vegetative stage of in vitro plantlets were enriched to a greater degree than in the reproductive stage. In contrast, the net photosynthetic rate (P n) of the plantlets derived from nodal explants was greater than that of plantlets derived from seedlings by a factor of 1.4 (Table 2), relative to sugar accumulation in the leaf tissues. Soluble sugars (including glucose, fructose, and sucrose) in the plantlets derived from nodal explants were manifestly enriched and at higher levels than in plantlets derived from seedlings (Fig. 3). Accumulated soluble sugars in the leaf tissues, major carbohydrate resource obtained from elevated photosynthetic rate and sucrose-applied medium, may play a key role in carbohydrate signaling for flowering initiation in rice plants. Also, P n in the flowering stage was elevated when compared to the vegetative stage. In vitro inflorescence production was not only successfully induced in the PT1 rice cultivar (SD photoperiod insensitive), but also in KDML105 (SD photoperiod sensitive), as demonstrated in Fig. 4. In the present study, rice inflorescences were successfully elicited, although in vitro conditions promoting floral blooming, fertilization, and seed set in plant tissue culture are still to be determined. The environmental conditions of in vitro culture are quite different from those of a natural paddy field. As a consequence, control of plant tissue culture environment will be further undertaken to study floral emergence, pollen viability, in vitro fertilization, and seed development.

Table 1. Flowering percentage (FP), number of flowers per cluster (FN), plant height (PH), number of leaves per plant (LN), leaf area (LA), fresh weight (FW), and dry weight (DW) of plantlets derived from seedlings (without flowering), or nodal explants (with flowering) cultured in modified MS medium for 90 d
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Figure 2.
figure 2

Morphological characteristics of PT1 plantlets derived from seedlings without flowering (A), or nodal explants with flowering (B; arrow) after culture in modified MS medium for 90 d.

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Table 2. Chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (TC), total carotenoid (C x+c ) content, and net photosynthetic rate (P n) of plantlets derived from seedlings (without flowering) or nodal explants (with flowering) cultured in modified MS medium for 90 d
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Figure 3.
figure 3

Glucose, fructose, and sucrose contents in the leaf tissues of PT1 plantlets derived from seedlings or nodal explants after culture in modified MS medium for 90 d. Different letters in each bar show a significant difference at p ≤ 0.01.

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Figure 4.
figure 4

In vitro flowering of KDML105 (O. sativa L. spp. indica cv. KDML105); arrows indicate the booting (A) and flowering stages (B) after culture in modified MS medium for 90 d.

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Discussion

A key factor for in vitro flowering in rice is the use of nodal explants from the booting stage in natural conditions as the initial plant material. In the case of the explant material, nodal segments from plant species have been used effectively for inflorescence development in the in vitro culture system. In addition, plant height and leaf size of in vitro plantlets were controlled using 6.81 μM paclobutrazol or anti-gibberellic acid in the culture medium. In previous reports, the function of paclobutrazol as a growth retardant to produce a compact or dwarf form (Thompson et al. 2005; Ghosh et al. 2010), and alternatively to regulate the floral organs of plants (Qiao et al. 2009; Wang et al. 2009) has been demonstrated. For example, the proportion of in vitro flowering of Saposhnikovia divaricata was 26% in a culture medium supplemented with 1.02 μM paclobutrazol, while plant height was retarded to 50% of the control (Qiao et al. 2009). Also, the proportion of in vitro flowering of the orchid species Dendrobium nobile was 33.3% in the culture medium containing 1.72 μM paclobutrazol (Wang et al. 2009). In the present study, plant height of in vitro plantlets was consequently controlled by adding paclobutrazol to the medium.

In the present study, the levels of photosynthetic pigments (including Chla, Chlb, TC, and C x+c ) in plantlets with no inflorescences (seedlings as initial explants) were higher than for flowering plantlets (booting nodal explants). In the reproductive stage of rice, photosynthetic pigments in the leaf tissues were generally reduced, depending on the number of days after flowering, leading to low P n (Murchie et al. 2002; Jiao et al. 2003; Zhang et al. 2007; Kumagai et al. 2009). In contrast, P n in this study was higher in the flowering plantlets in comparison to those with an absence of flowering. During in vitro culture, the major source of carbon is sugar in the culture medium (4% w/v sucrose), which is uptaken, translocated, and accumulated in the sink organs. So, the low level of P n in vegetative plantlets may derived from sugar-feedback inhibition. Floral development in rice relies on direct access to the carbon sources from the leaf factory tissues, which are enriched with soluble sugars. In floral initiation, sugar may play a key role in transition signaling, and was hence applied exogenously in Perilla frutescens (Zhang 2007), Spathiphyllum (Dewir et al. 2007), and K. leucocephala (Taylor et al. 2007).

In conclusion, inflorescences of in vitro cultured rice were successfully initiated using nodal explants derived from the booting stage of plants in pot culture. A dwarf plantlet was obtained in vitro by control treatment with paclobutrazol. Glucose was enriched in the leaf tissues of flowering plantlets as a dominant soluble sugar, which was demonstrated as a key factor for flower initiation and development in the rice crop. This innovative protocol was effectively demonstrated for KDML 105, a SD photoperiod-sensitive rice cultivar, which naturally differs from the SD photoperiod-insensitive variety PT1.