Plant Cell, Tissue and Organ Culture 78: 217–223, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
217
Effect of genotype and explant age on callus induction and subsequent plant regeneration from root-derived callus of Indica rice genotypes Md. Enamul Hoque1 & John W. Mansfield2,∗ 1 Biotechnology
Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh; 2 Department of Agriculture Sciences, Imperial College London, Wye campus, Ashford, Kent, TN25 5AH, UK (∗ requests for offprints: Fax: +44-20-7594-2640; E-mail:
[email protected])
Received 1 August 2003; accepted in revised form 10 December 2003
Key words: callus induction, genotypic variations, Indica rice, plant regeneration, root segments
Abstract An attempt has been taken to establish an efficient plant regeneration system in vitro from 3, 5, 7 and 9-days-old root segments of four Indica (Bangladeshi) rice genotypes. Genotypic effects were observed in callus induction and subsequent plant regeneration. Moreover, the stage of development of the root explants also played a significant role in callus formation and subsequent plant regeneration. Younger explants were more efficient in both callus induction and plant regeneration. Plants regenerated in vitro were successfully established in soil and produced fertile seeds. Abbreviations: 2,4-D – 2,4-dichlorophenoxyacetic acid; BAP – 6-benzylaminopurine; Kn – kinetin; NAA – αnaphthalene acetic acid Introduction The successful application of available genetic transformation methods in rice is possible when efficient and reproducible plant regeneration protocols are available for the particular cultivar (Jain, 1997). Therefore, the identification and screening of useful cultivars for callus growth and plant regeneration in vitro are prerequisites in genetic improvement programs (Abe and Futsuhara, 1986). Success in tissue culture and plant regeneration commonly depends on plant genotype, source and the developmental stage of the explant used, in addition to culture conditions (Jain, 1997). As genotypic effects are unavoidable, any strategy to improve plant regeneration must involve the use of appropriate explants for initiation of callus cultures and manipulation of culture conditions. For rice genetic transformation studies, embryogenic callus culture and subsequent plantlet regeneration are essential requirements. The major problems
of the culture in vitro of Indica rice are the low rate of callus production, somatic embryogenesis and subsequent plantlet regeneration (Chu and Croughan, 1990). Moreover, significant differences have been found among the different genotypes of Indica rice (Peng and Hodges, 1989). The origin, physiological state, age of the explant and the degree of differentiation of tissues have been identified as the main factors influencing regeneration through somatic embryogenesis. In general, it has been found that immature organs and meristematic tissues, which contain undifferentiated cells, are more suitable for plant regeneration than mature organs (Morrish et al., 1987). In rice, immature embryos were found to be the most responsive explants for tissue culture (Rueb et al., 1994). Since this explant source is restricted to a short period of the growth cycle of the rice plant, other explants such as mature embryos, leaves and roots, which are available throughout the year, are more suitable from the logistical point of view for rice tissue culture, provided that a high frequency of regeneration
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Figure 1. Showing the different steps from callus induction to maturity in rice root culture; (A) in vitro grown rice seedling for root explants from rice seed (a-3, b-5, c-7 and d-9-days-old seedling), (B) callus induction from root explants (4-weeks-old culture callus), (C) embryogenic callus from rice root explants (8-weeks-old), (D) SEM showing early formation of somatic embryos (arrowed, ×3400), (E) plants regenerated from root-derived calli (a – green plants and b – albino plants) and (F) root callus-derived mature rice plants.
can be achieved (Rueb et al., 1994). Therefore the purpose of the study described in this paper was to attempt to develop a reproducible plant regeneration system in vitro from root explants of selected Indica rice genotypes from Bangladesh for future genetic transformation studies.
Materials and methods Plant material Four rice genotypes (BR22, BRRI Dhan 29, BR584215-4-8 and Moulata) were used for this study. Excised
219 seedling roots were the source of in vitro experimental materials. Preparation of explants Mature healthy seeds were dehusked manually and then soaked in 70% (v/v) ethanol for 3 min with gentle agitation followed by rinsing three times with sterile distilled water. After that, the seeds were surface sterilized in 50% (v/v) commercial bleach (approximately 5% NaOCl) for 30 min with agitation, then rinsed with sterile distilled water five times. One surfacesterilized seed was aseptically inoculated into each glass tube (10 cm × 2.5 cm diameter) containing 10 ml Murashige and Skoog (1962) basal medium without added hormones, gelled with 0.3% (w/v) Phytagel and supplemented with 3.0% (w/v) sucrose. The cultures were then kept at 25 ± 1 ◦ C under a 16/8 h light/dark photoperiod regime provided by cool-white fluorescent light for raising seedling. Roots were cut aseptically into approximately 1 cm sections (not included root tips) at 3, 5, 7 and 9-days after seed was placed on the medium (Figure 1A). These root segments were used as explants for callus induction. Medium for callus initiation and maintenance Media for callus initiation and maintenance were based on the formulation of Murashige and Skoog (1962) and Linsmaier and Skoog (1965), gelled with 0.3% (w/v) Phytagel supplemented with 3% (w/v) sucrose and 2.0 mg l−1 2,4-D (designated as MSII and LSII, respectively). The media were freshly prepared, autoclaved and dispensed as 25 ml aliquots into 9 cm diameter Petri dishes. Callus initiation from root segments Aseptically cut root segments were placed onto callus induction media with eight segments in each Petri dishes (Figure 1B). The plates were then sealed with Nescoflim and incubated in the dark at 29 ± 1 ◦ C. After 4 weeks, calli derived from individual root segments were separated from the explant tissue and transferred to fresh medium, which had the same composition as that used for callus induction. Subculturing was then carried out at 2-week intervals with transfer of only the vigorously growing portions of calli. Cultures showing browning were discarded at each subculture.
Determination of callus induction ability Root segments were examined 4 weeks after plating onto callus induction media. Production of callus of any size was considered a positive result. Eight root segments were plated in each Petri dish and two Petri dishes were used per replication for each treatment in each genotype. The callus induction ability of each genotype was calculated as follows: Callus induction ability(%) Total number of root segments producing callus = × Total number of root segments cultured ×100.
Composition of regeneration medium (RM) Two regeneration media were used, they were designated as RMI and RMII. In both, MS salts were used as a basal component of the media tested. The only difference between the two media was in hormonal combination: RMI medium contained 2 mg l−1 BAP, 1 mg l−1 NAA and 1 mg l−1 Kn and RMII medium contained 2 mg l−1 BAP, 0.5 mg l−1 NAA. Sucrose was added to both the media at 30 g l−1 . Routinely, 10 ml molten medium gelled with 0.3% Phytagel (w/v) were dispensed into glass tubes (10 cm × 2.5 cm diameter) and the tubes were covered with a metal cap before autoclaving. The pH of the medium was adjusted to 5.8 before sterilization. Plant regeneration Eight-weeks-old only embryogenic calli (Figure 1C) were placed into glass tubes (10 cm × 2.5 cm diameter), containing 10 ml RM. Calli produced from the two different induction media (MSII and LSII) were transferred onto two different regeneration media (RMI and RMII) to examine the influence of callus induction media on subsequent regeneration frequencies. Cultures were kept at 25 ± 1 ◦ C under a 16-h photoperiod regime giving irradiance levels in the range of 55–62 µmol m−2 s−1 at the bench surface. In total, 16 tubes were used per replication for each treatment for each genotype. Data on plantlet regeneration were collected 6 weeks after transfer of calli. Green and albino plant regeneration frequencies were counted separately. The regeneration response of each genotype was calculated as follows: Plant regeneration response(%) Number of calli producing plants = × Total number of calli on the regeneration media ×100.
220 Table 1. Callus induction from root explants after culture for 4 weeks Genotype
Media
Percent callus induction (mean ± SE) from roots aged 3–9 days 3-days
5-days
7-days
9-days
Moulata
MSII LSII
96.0 ± 2.3 96.0 ± 0.0
82.7 ± 3.5 80.0 ± 6.1
73.3 ± 9.3 80.0 ± 8.3
73.3 ± 5.8 66.7 ± 4.8
BR5842-15-4-8
MSII LSII
94.7 ± 2.7 94.7 ± 2.7
89.3 ± 3.5 81.3 ± 2.7
84.0 ± 4.6 73.3 ± 5.3
81.3 ± 4.8 80.0 ± 4.0
BR22
MSII LSII
89.3 ± 2.7 77.3 ± 1.3
78.7 ± 4.8 74.7 ± 4.8
73.3 ± 2.7 65.3 ± 2.7
58.7 ± 4.8 60.0 ± 4.6
BRRI Dhan 29
MSII LSII
88.0 ± 6.1 93.3 ± 4.8
84.0 ± 4.6 81.3 ± 2.7
66.7 ± 4.8 78.7 ± 5.3
76.0 ± 9.2 80.0 ± 2.3
LSD (5%) = 13.55. Data were taken from average of three replicate and each replication has 16 root explants.
Table 2. Plant regeneration from callus produced in MSII induction medium from root explants of different age Genotype
Media
Percent plant regeneration (mean ± SE) from 3, 5, 7 and 9-days-old root-derived calli 3-days
5-days
Green
Albino
Green
7-days Albino
9-days
Green
Albino
Green
Albino
16.7 ± 4.2 8.3 ± 4.2
16.7 ± 4.2 16.7 ± 4.2
BR5842-15-4-8
RMI RMII
37.5 ± 14.4 25.0 ± 14.4
12.5 ± 7.2 37.5 ± 14.4
8.3 ± 4.2 16.7 ± 4.2
4.2 ± 4.2 8.3 ± 8.3
16.7 ± 11.0 20.8 ± 11.0
0.0 16.7 ± 4.2
BR22
RMI RMII
33.3 ± 15.0 37.5 ± 7.2
29.2 ± 15.0 20.8 ± 11.0
25.0 ± 12.5 20.8 ± 11.0
8.3 ± 8.3 16.7 ± 11.0
16.7 ± 8.3 4.2 ± 4.2
4.2 ± 4.2 0.0
0.0 0.0
0.0 0.0
BRRI Dhan 29
RMI RMII
4.2 ± 4.2 8.3 ± 8.3
4.2 ± 4.2 0.0
4.2 ± 4.2 0.0
4.2 ± 4.2 0.0
12.5 ± 7.2 20.8 ± 4.2
0.0 0.0
0.0 0.0
0.0 0.0
Data were taken from average of three replicate and each replication contained 16 calli. Moulata failed to produce any plants.
Table 3. Plant regeneration from callus produced in LSII induction medium from root explants of different age Genotype
Media
Percent plant regeneration (mean ± SE) from 3, 5, 7 and 9-days-old root-derived calli 3-days
5-days
7-days
9-days
Green
Albino
Green
Albino
Green
Albino
Green
Albino
16.7 ± 4.2 12.5 ± 0.0
25.0 ± 14.4 4.2 ± 4.2
BR5842-15-4-8
RMI RMII
25.0 ± 7.2 25.0 ± 0.0
12.5 ± 7.2 25.0 ± 7.2
20.8 ± 8.3 20.8 ± 15.0
4.2 ± 4.2 16.7 ± 11.0
12.5 ± 0.0 8.3 ± 8.3
8.3 ± 8.3 4.2 ± 4.2
BR22
RMI RMII
20.8 ± 11.0 8.3 ± 8.3
25.0 ± 14.4 0.0
12.5 ± 12.5 4.2 ± 4.2
8.3 ± 8.3 4.2 ± 4.2
4.2 ± 4.2 0.0
4.2 ± 4.2 0.0
0.0 0.0
0.0 4.2 ± 4.2
BRRI Dhan 29
RMI RMII
4.2 ± 4.2 0.0
0.0 4.2 ± 4.2
0.0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
4.2 ± 4.2 8.3 ± 8.3
0.0 0.0
Data were taken from average of three replicate and each replication contained 16 calli. Moulata failed to produce any plants.
221 Scanning electron microscopy (SEM) For SEM, 8-weeks-old embryogenic callus pieces were taken from the callus culture. Callus pieces were fixed overnight with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) at 8 ◦ C, washed twice in the same buffer and dehydrated through a series of acetone solutions (50, 70, 80 and 90% v/v) for 1 h each. The final dehydration was in three changes of 100% acetone for 1 h. After that, the dehydrated specimens were critical point dried, and then dried specimens were mounted on aluminium stubs using epoxy resin based Araldite glue. Finally, the mounted specimens were coated with a very thin layer of gold, and subsequently the coated specimens were examined in a scanning electron microscope and photographs were taken where necessary. Experimental design and statistical analysis A completely randomized with three replications per treatment for each genotype were used in this study. Analysis of variance (ANOVA) was done only on the basis of genotype, explant age and callus induction media. In the plant regeneration experiment, due to predominance of zeros, an ANOVA was not applicable; only means and standard errors of means (SE) were calculated.
Results Callus induction Callus tissues were initiated from root segments on both the MSII and LSII induction media supplemented with 2 mg l−1 2,4-D. The initiation of callus started 5–7 days after inoculation from the cut ends of root segments and after 14 days from the entire length of root segments. However, some root segments only produced callus from few sites in the segments. Initially, callus tissues were highly mucilaginous, soft and covered with a translucent sticky substance. Four weeks after induction, calli were removed from the root segments and transferred onto fresh media. Following this subculture, white to pale-yellow embryogenic calli appeared on the surface of the mucilaginous callus. Callus induction was significantly affected by the rice genotype (p > 0.001) as well as the age of the root segments (p > 0.001) but not significantly affected by the callus induction media (p = 0.326). Three
and five-days-old root segments produced significantly more callus than those of 7 and 9-days-old segments. However, the highest rate of callus induction was found from 3-days-old root segments irrespective of rice genotypes used in this experiment. Among the four rice genotypes, Moulata had the highest frequency of callus production both on MSII and LSII induction media (Table 1). Plant regeneration Following transfer of the embryogenic calli onto regeneration media, green areas began to develop within 2–3 weeks, and then green plants (with a shoot and root axis) developed from those green areas (Figure 1Ea). At the same time some albino plants also developed from various calli (Figure 1Eb). Some pieces of callus produced both green and albino plants and others produced only one type of plant. On average, regeneration of green plants was higher from calli produced on MSII callus induction medium (Tables 2 and 3) except in genotype BR5842-15-4-8 with 5-days-old root calli (on both RMI and RMII regeneration media) and with 9-days-old root calli (only for RMII media). In vitro regenerated green plants were established in soil and produced fertile seeds (Figure 1F). Regeneration responses were found to be affected by the genotype as well as age of initial root segments. Only the rice genotype BR5842-15-4-8 produced green plants with all four ages of root-derived calli. On the other hand, Moulata did not produce any plants at all. In relation to regeneration response, BR5842-15-4-8 was found to be the best genotype followed by BR22 and BRRI Dhan 29. Irrespective of callus induction medium, RM and genotype, 3-daysold root segments gave the best regeneration of green plants. SEM Scanning electron micrographs showed the early formation of somatic embryos in each genotype examined (Figure 1D), from which simultaneous appearance of shoots and roots were noticed after transferred in regeneration media (Figure 1E).
Discussion A reproducible system for plant regeneration via somatic embryogenesis was developed for some
222 Bangladeshi rice (Indica) genotypes from root explants. Plant regeneration from rice root cultures was first reported by Kawata and Ishihara (1968). They mentioned that the type of plant regeneration was organogenesis since no embryo-like structure could be found. However, a subsequent investigation with rice root cultures (Abe and Fustuhara, 1984) showed that two different pathways for plant regeneration, that is, organogenesis and somatic embryogenesis, could both be found. Later, it was confirmed that rice root cultures easily formed embryogenic callus, which readily gave rise to somatic embryos that germinated into plantlets (Abe and Fustuhara, 1985). From the present studies, SEM observations demonstrated the early formation of rice somatic embryos (Figure 1D) in callus culture, and the simultaneous formation of shoots and roots from those somatic embryos after transferred into RM, with a direct connection of shoot and root axis, suggesting that plants regenerated through somatic embryogenesis (Chowdhry et al., 1993; Basu et al., 1997). Significant genotypic variation in callus induction and plant regeneration potential have been reported in rice (Abe and Fustuhara, 1986; Hartke and Lörz, 1989; Khanna and Raina, 1998; Lee et al., 1999). In this study, it was also found that there was a significant difference (p < 0.001) in callus induction between the four genotypes. Three-days-old root segments produced significantly more calli (p < 0.001) and subsequently more green plantlets than those of 5, 7, and 9-days-old root segments. Our findings are, therefore, in agreement with the proposed by Vasil and Vasil (1986) that the developmental stage of the explant plays an important role in embryogenic callus induction and plantlet regeneration. In barley, similar results were found using differently aged root explants for callus induction and plant regeneration (Chand and Sahrawat, 2000). Initially, root callus tissues were highly mucilaginous, soft and covered with a translucent sticky substance. But after subculture, white to pale-white embryogenic calli appeared on the surface of the mucilaginous callus. This observation is similar to that reported by Abe and Futsuhara (1985). Initiation of callus was predominantly from cut ends of the root segments. Out of four genotypes, Moulata failed to produce plants from callus and the other three had differing degrees of success. Similarly, Hartke and Lörz (1989) found that out of 15 Indica rice varieties tested, seven produced embryogenic calli, but only four regenerated plants.
Regeneration of albino plants was frequently observed in the present study. Wang et al. (1978) mentioned that the number of albino regenerants increased when the temperature was elevated above the normal growth temperature for callus induction. In rice tissue culture, around 25 ◦ C temperature is widely used for callus induction (Adkins, 1992; Kunanuvatchaidach et al., 1995). However, in the present study, 29 ± 1 ◦ C was used for callus induction from rice root explants and the higher temperature might have increased the frequency of albino plants. In order to obtain high totipotency, suitable genotypes and appropriate tissue sources should be utilized. From our studies it may be concluded that 3-days-old root segments were the best for the induction of embryogenic callus and green plant regeneration using Indica rice genotypes from Bangladesh. Such type of studies may be useful in genetic transformation experiments where objective is to obtain fertile transgenic plants from the specific tissues.
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