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A.R. Zuraida, A.S. Zulkifli , H. Habibuddin and B. NaziahJ. Trop. Agric. and Fd. Sc. 39(2)(2012): 167– 177

Regeneration of Malaysian rice variety MR 219 via somatic embryogenesis(Regenerasi pokok padi Malaysia varieti MR 219 melalui prosesembriogenesis somatik)

A.R. Zuraida*, A.S. Zulkifli*, H. Habibuddin** and B. Naziah*

Keywords: ABA, 2,4-D, NAA, Oryza sativa ssp. indica, plantlet regeneration, somatic embryogenesis, tissue culture, rice variety MR 219

AbstractTissue culture plant regeneration protocols were developed for the Malaysian rice variety MR 219 (Oryza sativa L. ssp. indica). Following these protocols, 78% of callus became embryogenic when cultured on a medium containing 1 mg/litre 2,4-D (2,4-dichlorophenoxyacetic acid) and 10 mg/litre NAA (a-naphthaleneacetic acid). The highest frequency of somatic embryo initiation (81%) was obtained by incubating embryogenic calli on a medium containing 10 mg/litre ABA (abscisic acid) and 9 g/litre gelrite agar for 6 weeks. These embryos regenerated the highest number of plantlets when they were transferred to a medium containing 3 mg/litre kinetin and 0.5 mg/litre NAA. After 8 weeks incubation, 9 plantlets per 3 g of somatic embryos were produced. Hence, by manipulating plant growth regulators in the culture media, one medium was established for each of the following phases, namely the induction of embryogenic calli, their subculture and the regeneration of plantlets. These protocols can facilitate the production of large numbers of embryogenic calli with high regeneration capacity, and maybe incorporated into the genetic transformation system for the rice variety MR 219.

*Biotechnology Research Centre, MARDI Headquarters, Serdang, P.O. Box 12301, 50774 Kuala Lumpur, Malaysia**Rice and Industrial Crops Research Centre, MARDI Headquarters, Serdang, P.O. Box 12301, 50774 Kuala Lumpur, MalaysiaAuthors’ full names: Zuraida Ab. Rahman, Zulkifli Ahmad Seman, Habibuddin Hashim and Naziah Basirun E-mail: [email protected]©Malaysian Agricultural Research and Development Institute 2011

IntroductionThe commercial Malaysian Indica rice variety, MR 219, was the first rice variety to be developed by the Malaysian Agricultural Research and Development Institute (MARDI) based on a direct seeding planting system (Alias 2002). This rice variety has been classified as high-yielding, producing more than 10 mt/ha. Grain weight can be as high as 28–30 mg, and as many as 200 grains/panicle can be obtained, this being higher than the number from rice

varieties previously released by MARDI (Alias 2002). Other good characteristics of this variety include a short maturation period of 105–111 days, fairly tall but strong culms, and resistance to blast and bacterial leaf blight. The rice is commonly marketed as a long-grain variety (Alias 2002). The productivity of MR 219 and other varieties of rice can be further improved in many ways. Among the promising approaches applicable to rice breeding is transgenic technology, which has become

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Regeneration of rice variety MR 219

a powerful tool in crop improvement. Research targets that previously lay outside the reach of traditional plant breeding and genetics can now be realized through transgenic techniques. Genetic manipulations that benefit from transgenesis include the introduction of homologous or heterologous genes into plant genomes to modify the functions of genes and alter their expression patterns (Wei et al. 2009). Genetic modification through transgenic technology commonly involves an in vitro culture stage. Plantlet regeneration in tissue culture is therefore an important component in many biotechnological tools for genetic improvement (Santacruz-Ruvalcaba et al. 1998). Successful genetic transformation in rice is possible only when efficient and reproducible plant regeneration protocols are available for the particular cultivars. Several tissue culture methods for different rice varieties have been reported (Aldemita and Hodges 1996; Rashid et al. 1996; Khanna and Raina 1998; Zuraida et al. 2010). With some varieties, however, the lack of an efficient plantlet regeneration system poses a serious bottleneck to the application of genetic transformation technology. Taking cognizance of in vitro culture being a prerequisite for the genetic transformation of rice, MARDI has recently succeeded in obtaining up to 97% somatic embryogenesis in callus developed from mature seeds of the rice variety MR 232 using a culture medium containing a combination of 1 mg/litre 2,4-D and 5 mg/litre NAA (Zuraida et al. 2010). Syaiful et al. (2009) was able to produce 80% embryogenesis in the callus of MR 219 rice variety using a modified MS culture medium containing 4 mg/litre of 2,4-D. The next principal objective in the rice tissue culture programme is the development of a plantlet regeneration system for the commercial Malaysian indica rice variety MR 219 through somatic embryogenesis. In this paper, we present the results of our study on the production of embryogenic calli of MR 219 and the subsequent

development of somatic embryos on MS medium modified with 2,4-D and NAA. The effects of plant growth regulators on the rate of production of MR 219 embryogenic calli on modified MS and subsequent plantlet regeneration are also discussed. The successful development of a rice plantlet regeneration system sets the stage for further improvement of the Malaysian indica rice variety through genetic engineering.

Materials and methodsSeed sterilizationSeeds of the rice variety MR 219 were dehulled, and sterilized by immersion in 50–100% ethanol for 2 min, then in 1% Vircon for 30 min. Vircon is a multi-purpose disinfectant containing potassium peroxymonosulphate, sodium dodecylbenzenesulfonate, sulphamic acid and inorganic buffers. It is typically used for cleaning up hazardous spills, disinfecting surfaces and soaking. The seeds were then rinsed three times in sterile distilled water, followed by immersion in 50–100% Clorox (sodium hypochlorite, 5.2%), supplemented with or without 2 drops of Tween 20 for 40 min. Tween 20 is a polysorbate surfactant whose stability and relative non-toxicity allows it to be used as a detergent and emulsifier in tissue culture applications. After rinsing three times in sterile distilled water, the seeds were cultured on the specified callus induction media and incubated in the dark for 2 weeks at 26 ± 2 °C. The cultured seeds were then observed after 2 weeks and the percentage of survivals recorded. The 12 different treatments used for sterilization of dehulled seeds are listed in Table 1.

Callus inductionSterilized seeds were plated aseptically on solidified media containing MS basal medium (Murashige and Skoog 1962), inorganic salts, 3% sucrose, 0.3% gelrite agar and different concentrations of NAA (0–50 mg/litre) or 2,4-D (0–50 mg/


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A.R. Zuraida, A.S. Zulkifli , H. Habibuddin and B. Naziah

Table 1. Sterilization steps of MR 219 rice seeds

Treat.No.

Step 1(2 min)

Step 2(30 min)

Step 3(1 min)

Step 4(40 min)

Step 5(1 min)

1 Ethanol 50%

Vircon 1% Rinse 3x with distilled water

Clorox 50%

Rinse 3x with distilled water

2 Ethanol 50% Clorox 80% 3 Ethanol 50% Clorox 100% 4 Ethanol 50% Clorox 50%+Tween 20 5 Ethanol 50% Clorok 80%+ Tween 20 6 Ethanol 50% Clorox 100%+ Tween 20 7 Ethanol 100% Clorox 50% 8 Ethanol 100% Clorox 80% 9 Ethanol 100% Clorox 100%10 Ethanol 100% Clorox 50%+ Tween 2011 Ethanol 100% Clorok 80%+ Tween 2012 Ethanol 100% Clorox 100%+ Tween 20

litre). The pH of the media was adjusted to 5.7– 5.8 before autoclaving. Cultured seeds were maintained in the dark room at 25–27 °C for 30 days. Calli were produced after 3–4 weeks and recorded as percentage of callus induction. The calli were then transferred (sub-cultured) onto fresh media of the same composition. The percentage of callus browning response was recorded after 3–4 weeks. The experiment was repeated using different hormone combinations of NAA (1–50 mg/litre) and 2,4-D (1–10 mg/ litre), and the frequency of embryogenic callus development recorded after 6 weeks of culture.

Somatic embryogenesisHigh quality embryogenic calli were selected and transferred to MS media containing ABA (1, 5 and 10 mg/litre) in different concentrations of agar (3, 6, 9, 12 and 15 g/litre). The cultures were then incubated for 2, 4, 6 or 8 weeks, and the percentage formation of whitish embryos was recorded. A sample (3 g) of the whitish embryos produced was then cultured on MS basal medium with or without additional plant growth regulators (3 mg/litre kinetin and 0.5 mg/litre NAA) for further development. The number of regenerated plantlets was recorded after 4–8 weeks.

Statistical analysisThe experiments were arranged in a completely randomized design (CRD) as single factor experiments with four replications, with each replication per treatment containing ten explants. All experiments were repeated three times. Analysis of variance was conducted on the data and the least significance difference (LSD) was used as the test of significance.

Results and discussionSeed sterilizationThe efficiency of different sterilisation techniques on survival of dehulled seeds was studied (Table 1). Generally, seeds immersed in 100% Clorox showed a higher survival rate (73–85%), whereas seeds sterilised with 50% Clorox did not survive (Figure 1). Survival rate was improved when Tween 20 was added in the treatment, but different ethanol concentrations did not show any significant effect. The most effective sterilization technique for callus development was treatment 12 (Table 1), which attained an 85% survival rate (Figure 1). With this procedure, the dehulled seeds were immersed in absolute ethanol (2 min), followed by 1% Vircon (30 min) and rinsing three times in sterile distilled water before further immersion in 100% Clorox


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supplemented with 2 drops of Tween 20 for 40 min. The seeds were rinsed 3 times with sterile distilled water before use in callus induction experiments.

Callus inductionSeeds were cultured on callus induction medium to evaluate the effects of different concentrations of 2,4-D or NAA on callus induction. Callus formation varied among the treatments tested, as shown in Table 2. In general, the rate of callus formation varied depending on the concentration of hormone used. Media containing 2,4-D alone showed the earliest signs of callus formation. The callus was developed from the scutellar region of the seed, and was visible after 14 days. The primary callus proliferated from yellowish to white tissue after 3–4 weeks. Culture media supplemented with NAA alone or control (no hormone) showed no callus development, but the seeds germinated rapidly into plantlets. The highest percentage of callus induction (85%) was obtained on media containing 5 mg/litre 2,4-D, followed by 1 mg/litre 2,4-D (80%) and 10 mg/litre 2,4- D (67%) (Table 2). At this stage, the callus was transferred onto fresh medium of the same composition for 3–4 weeks

0

10

20

30

40

50

60

70

80

90

1 2 3 4 5 6 7 8 9 10 11 12

Treatments

Surv

ives

(%)

Figure 1. Efficiency of different sterilization techniques on survival of dehulled MR 219 seeds

for callus multiplication and to prevent browning. Callus browning was commonly seen in untransferred callus remaining on the same medium. Supplementation with increasing concentrations of 2,4–D resulted in a higher incidence of browning, ranging from 56% to 100% (Table 3). Even though compact and globular calli were obtained by using 2,4-D, only some calli could proliferate. Others turned brown and watery when continually sub-cultured on the same medium. No callus formation was observed in the absence of 2,4-D in the culture medium. Vikrant and Rashid (2003) considered 2,4-D to be the most reliable growth regulator for the induction of the somatic embryogenesis. This view is consistent with our observations on callus induction. According to Shahsavari et al. (2010), application of 2 mg/litre 2,4-D gave rise to the highest callusing frequency on the Malaysian upland rice cultivar. In another study, Tohru (1989) found that prolonged culture led to the browning of callus tissue, especially on media containing 2,4-D. He suggested that month-old cultures on primary media with 2,4-D be transplanted to medium containing NAA or BA to prevent callus browning. The effect of combining


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Table 4. Effects of different combinations of NAA and 2,4-D concentrations on percentage of callus induction

2,4-D(mg/litre)

NAA (mg/litre)

1 5 10 20 50 1 54 ± 10 67 ± 9 79 ± 9 68 ± 12 18 ± 3 5 31 ± 5 34 ± 6 59 ± 8 55 ± 6 45 ± 910 11 ± 1.1 16 ± 0.9 17 ± 5 8 ± 0.5 0

Plate 1. Embryogenic callus on medium supplement with 1 mg/litre 2,4-D and10 mg/litre NAA

Table 2. Percentage of callus induction of MR 219 seeds cultured on MS media and supplemented with different concentrations of NAA or 2,4-D

Concentration of hormone 0 1 5 10 20 50(mg/litre)

NAA 0 0 0 0 0 02,4-D 0 80 ± 12 85 ± 13 67 ± 9.8 55 ± 9 15 ± 1.4

Table 3. Percentage of browning response of MR 219 callus one month after second sub-culture on callus induction medium supplemented with different concentrations of NAA or 2,4-D

Concentration of hormone 0 1 5 10 20 50(mg/litre)NAA – – – – – –2,4-D – 56 ± 4 67 ± 7 78 ± 10 96 ± 14 100 ± 18– = No callus

2,4-D with NAA on callus formation was hence evaluated. The effect of various combinations of 2,4-D and NAA on callus induction is shown in Table 4. After a month, callus formation, ranging from 8 to 79%, was

observed in all the combinations of the two growth regulators. Callus growth on media with both growth regulators showed faster growth and proliferation than those cultured in medium containing 2,4-D alone. The highest rate of callus formation (79%) was observed on the medium containing a combination of 1 mg/litre 2,4-D and 10 mg/litre NAA. The texture of the callus surface was nodular, bearing a number of globular structures approximately 1 mm in diameter (Plate 1). On the other hand, the surface of callus cultured on medium with 2,4-D alone was smooth. The addition of more than 5 mg/litre 2,4-D or 20 mg/litre NAA resulted in decreased callus formation. The incidence of the browning response in callus treated with different 2,4-D and NAA combinations is shown in Table 5. Calli cultured on media containing both 2,4-D and NAA gave less browning as compared with those treated with 2,4-D alone. The incidence of browning ranged


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from 0–65%. The browning effect was minimal when the combinations of 2,4- D and NAA were from 1–5 mg/litre and 5–20 mg/litre respectively. These calli were compact, globular, and yellowish; they tended to be variable in their propensity to proliferation. The frequency of embryogenic calli obtained is shown in Table 6. After 6 weeks of culture, the embryogenic calli were transferred to pre-regeneration medium to allow plantlets to develop. The frequency of embryogenic calli ranged from 14– 78% depending on the culture medium. Embryogenic calli were most frequently observed with the combination of 1 mg/litre 2,4-D and 10 mg/litre NAA (78%), followed by the combination 5 mg/litre 2,4-D and 10 mg/litre NAA (56%). The formation of soft, white, non-embryogenic calli was observed initially on the medium with low concentrations of 2,4-D alone, with most of the calli turning brown after subculture. These calli remained as a cell mass without developing into embryos, as also observed by Girijashankar (2007). Lee et al. (2002) found that the number, colour, size, shape and the appearance of the embryogenic calli varied among the rice genotypes, depending on the type of basal medium. This indicated

that induction of high-quality rice callus was influenced by genotype, medium, and the kind of explants as well as by their interactions. The induction of embryogenic calli in rice is considered as the most critical step for successful genetic plant transformation (Lee et al. 2002). In our study, the response of indica rice to embryogenic callus induction benefited from the presence of both 2,4-D and NAA. The use of 2,4-D alone would induce callusing very well, but such calli suffered from browning. The addition of NAA essentially overcomes this problem. Despite previous reports to the contrary (Swamynathan et al. 2010), a high frequency of somatic embryogenesis induction was obtained in the present study in response to the auxins NAA and 2,4-D, a development also noted by Magioli et al. (2001). Seetharama et al. (2000) has confirmed the role of 2,4-D in induction of callus in sorghum shoot apical meristems. Harshavardhan et al. (2002) reported that MS medium supplemented with BAP (2–4 mg/litre) and 2,4-D (0.5 mg/litre) always gave rise to a certain degree of callus formation, whereas replacement of 2,4-D with NAA resulted in the effective induction of somatic embryos without callus formation.

Table 6. Frequency of embryogenic calli (%) after 8 weeks on callus induction media supplemented with different combinations of NAA and 2,4-D

Concentration of hormone NAA(mg/litre)

2,4-D 1 5 10 20 50

1 18 ± 3 34 ± 4 78 ± 13 51 ± 7 21 ± 4 5 34 ± 6 45 ± 2.5 56 ± 7 31 ± 9 23 ± 510 14 ± 0.6 18 ± 7 21 ± 2 21 ± 6 19 ± 6

Table 5. Browning response (%) of calli one month after first sub-culture on callus induction media supplemented with different combinations of NAA and 2,4-D

Concentration of hormone NAA(mg/litre)

2,4-D 1 5 10 20 50

1 2 ± 0.05 0 0 0 23 ± 3 5 4 ± 0.1 0 0 0 34 ± 410 12 ± 0.9 16 ± 3 23 ± 6 34 ± 5 65 ± 11


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Somatic embryogenesisEmbryogenic calli were transferred onto pre-regeneration medium in order to investigate their efficiency of regeneration for genetic transformation studies planned for the future. The formation of whitish somatic embryos (Plate 2) induced from embryogenic calli was evaluated in media containing 1, 5 and 10 mg/litre ABA contained in 6 –15 g/litre agar. After an incubation period of 6 weeks, the best medium, which comprised of 10 mg/litre ABA in 9 g/litre agar, showed the highest rate of somatic embryo formation (81%) (Table 7). An agar concentration of 3 g/litre was too low to be effective for any concentration of ABA. Having determined that the combination of 6–9 g/litre agar, 5–10 mg/litre ABA and incubation for 4–8 weeks promoted good embryo formation, these parameters were retained in the following experiments to regenerate plantlets. A histological examination was made of the somatic embryos produced through the present protocol. The cells of the somatic embryos exhibited dense cytoplasm and a reduced number of vacuoles. They showed a high nucleoplasmic ratio and densely stained nucleoli (Plate 3). To determine their potential for regeneration, the somatic embryos were transferred to different culture media for further development into plantlets. The regeneration media were formulated with or without additional Plant Growth Regulators

(PGR), comprising 3 mg/litre kinetin and 0.5 mg/litre NAA. The number of green plantlets that were regenerated is shown in Tables 8a and 8b. We observed that somatic embryos on regeneration media with PGR produced much higher numbers of plants than embryos that were not supplied with PGR. After 2 months, the media

Plate 2. Whitish somatic embryos developing from embryogenic calli

Table 7. Formation of whitish embryos (%) derived from embryogenic calli

Agar (g) ABA 1 mg/litre ABA 5 mg/litre ABA 10 mg/litre

weeks weeks weeks2 4 6 8 2 4 6 8 2 4 6 8

3 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 41 56 34 9 0 0 13 21 0 0 54 37 0 75 81 8012 0 0 14 32 0 0 41 27 0 43 41 3415 0 0 5 8 0 0 14 17 0 34 21 20

Plate 3. Cross-section of somatic embryo


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Plate 5. Tissue culture plants in the greenhouse

Plate 4. Development of plantlets after two months of culture

containing PGR produced 0–9 plantlets/3 g embryos (Table 8b) as compared with the media lacking in PGR, which produced 0–4 plantlets/3g embryos (Table 8a). The highest number of regenerated plantlets was obtained using the culture medium containing PGR, and where the embryos had been pre-treated earlier with 10 mg/litre ABA in 9 g/litre agar for 8 weeks. The regenerated plantlets were sub-cultured to fresh regeneration media for a month for further proliferation and rooting. During sub-culture, the shoot further elongated and multiplied. Regenerated plantlets 2 months after culture under light are shown in Plate 4. After acclimatization, the plantlets were established, with a survival rate of more than 95% under greenhouse conditions (Plate 5).

Table 8. Number of rice plantlets/per 3 g of somatic embryos obtained after pre-treatment with ABA and cultured on regeneration medium

(a)Transfer onto MS basal (without PGR)Agar (g) ABA 5 mg/litre ABA 10 mg/litre weeks weeks 4 6 8 4 6 8 6 0 0 0 1 2 3 9 0 0 0 1 2 412 0 0 0 0 0 115 0 0 0 0 0 0

(b)Transfer onto MS basal (with PGR)

Agar (g) ABA 5 mg/litre ABA 10 mg/litre weeks weeks 4 6 8 4 6 8 6 0 2 2 2 7 7 9 0 1 3 1 6 912 0 0 3 1 6 615 0 0 2 1 3 3PGR = Plant Growth Regulators (3 mg/litre kinetin and 0.5 mg/litre NAA)

ABA is an important plant growth regulator involved in various physiological processes in plants (Zeevaart and Creelman 1988). ABA also plays an important role in vegetative development in response to various environmental osmotic stresses (Machuka et al. 1999; Xiong et al. 2002). Diah and Bhalla (2000) reported that no albino rice plantlet was obtained when ABA was applied, and that the treatment enabled the regenerated plantlet to withstand partial desiccation. Similar observations were made in the present study. The ABA treatment that improved plantlet regeneration had an effect similar to the partial desiccation of the embryogenic callus that leads to an increase in endogenous ABA level (Yang et al. 1999), and improvement of oxygen available to the calli (Jain and Wu 1996). According to Wallin and Johansson (1989), pre-


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Harshavardhan, D., Rani, T.S., Ugalanathan, K. and Seetharama, N. (2002). An improved protocol for regeneration of Sorghum bicolor from isolated shoot apices. Plant Biotechnology 19(3): 163–171

Jain, R.K.S. and Wu, R. (1996). Stimulatory effect of water stress on plant regeneration in aromatic Indica rice varieties. Plant Cell Rep. 15(6): 449–454

Khanna, H.K. and Raina, S.K. (1998). Agrobacterium-mediated transformation of indica rice cultivar using binary and superbinary vectors. Australian journal of Plant Physiology 26: 311–324

Lee, K., Jeon, H. and Kim, M. (2002). Optimization of mature embryobased in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from Japonica rice cultivars. Plant Cell Tissue Organ Cult. 71: 237–244

Machuka, J., Bashiardes, S., Ruben, E., Spooner, K., Cuming, A., Knight, C. and Cove, D. (1999). Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens. Plant Cell Physiol 40: 378–387

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treatment of the plant with ABA stimulated initial cell division. Plantlet regeneration frequency was three- to fourfold higher in the media containing 1 mg/litre ABA than in media lacking ABA (Saito et al. 1997). ABA is known to play an important role in physiological desiccation (Meurs et al. 1992). In producing regenerants from calli, there appeared to be a correlation between the desiccation of the medium and the presence of ABA. The plant growth regulators, kinetin and NAA, have been shown to be useful in plantlet regeneration in other studies. For example, MS media supplemented with 0.8 mg/litre NAA and 0.8 mg/litre kinetin induced axillary bud proliferation (Mungole et al. 2009). Rao and Singh (1991) cultivated leaf explants in medium supplemented with 0.5 and 10.8 μM NAA, and embryogenic calli were obtained in the presence of 27 and 64 μM NAA combined with 0.5 mM kinetin. Swamynathan et al. (2010) found that a combination of NAA and kinetin gave a very high rate (88%) of plantlet development.

ConclusionThe maximization of in vitro culture conditions, with the development of a tissue culture protocol and the choice of responsive rice genotypes, are important in plant improvement programmes. Tissue culture is requisite in the approach to breeding new varieties through genetic transformation. The protocols presented here were developed for the regeneration of embryogenic calli of MR 219 rice variety into plantlets. We have established that a combination of 1 mg/litre 2,4-D and 10 mg/litre NAA is optimal for the callus induction medium, while 10 mg/litre ABA and 9 g/litre gelrite agar is suggested for the somatic embryo and plantlet regeneration medium. This system can be incorporated into a genetic engineering programme to introduce desirable agronomic traits in Malaysian rice varieties.


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Syaiful, B.P., Siti, N.A.A., Maheran, A.A., Sariah, M. and Othman, O. (2009). Somatic embryogenesis from scutellar embryo of Oryza sativa L. var. MR 219. Pertanika Journal of Tropical Agriculture Science 32(2): 185–194

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Yang, Y.S., Zheng, Y.D., Chen, Y.L. and Jian, Y.Y. (1999). Improvement of plant regeneration from long term cultured calluses of Taipei 309, a model rice variety in in vitro studies. Plant Cell Tissue Organ Cult. 57(3): 199–206

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Zuraida, A.R., Suri, R., Wan Zaliha, W.S. and Sreeramanan, S. (2010). Regeneration Of Malaysian indica rice (Oryza sativa) variety MR 232 via optimised somatic embryogenesis system. Journal of Phytology 2(3): 30–38


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AbstrakSatu protokol kultur tisu bagi regenerasi pokok padi varieti MR 219 telah berjaya dibangunkan. Sebanyak 78% kalus berjaya bertukar menjadi kalus embriogenik apabila dikultur di atas medium yang mengandungi 1 mg/liter 2,4-D (asid 2,4-diklorofenoksiasetik) dan 10 mg/liter NAA (asid asetik a-naftalena). Peratusan embrio somatik yang paling tinggi (81%) diperoleh dengan pengeraman kalus embriogenik di atas medium yg mengandungi 10 mg/liter ABA dan 9 mg/liter agar gelrite selama 6 minggu. Hasil paling tinggi diperoleh apabila embrio dieram di atas medium mengandungi 3 mg/liter kinetin dan 0.5 mg/liter NAA. Selepas 8 minggu pengeraman pokok yang diregenerasi mengeluarkan 9 pokok anak tumbuhan bagi setiap 3 g embrio. Dengan itu, tiga medium berbeza bagi kultur tisu padi MR 219 untuk penghasilan kalus embriogenik, sub-kultur dan regenerasi berjaya dibangunkan melalui manipulasi hormon tumbesaran pertumbuhan ke atas kultur kalus padi. Protokol ini mampu menghasilkan dan memperbanyakkan bilangan kalus embriogenik dengan kapasiti regenerasi yang tinggi. Medium yang diperoleh ini juga boleh membantu meningkatkan kecekapan sistem transformasi padi MR 219.

Accepted for publication on 12 July 2011


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