quantitative and qualitative assessment of phenotypic ...

Indian J. Plant Physiol., 2003 (Special Issue) pp. 613-619

QUANTITATIVE AND QUALITATIVE ASSESSMENT OF PHENOTYPIC VARIATION IN OIL PALM (ELAEIS GUINEENSIS JACQ. TENERA) PLANTLETS REGENERATED FROM TRANSFORMED AND NON-TRANSFORMED CALLI C.L. TAN1, S.J. BHORE1 AND F.H. SHAH2* 1School

of Bioscience and Biotechnology, Faculty of Science and Technology, National University of Malaysia, 43600 Bangi, Selangor, Malaysia, 2Melaka Institute of Biotechnology, Ayer Keroh 75450, Melaka, Malaysia

SUMMARY Somaclonal variation is a major concern in micropropagation of oil palm. We report the effect of stress caused by particle bombardment and subsequent selection on phenotype of Elaeis guineensis Jacq. Tenera plantlets, which were regenerated through somatic embryogenesis from transformed and non-transformed calli. The highest phenotypic abnormality displayed was 40.4% in plantlet population (P3) regenerated from transformed calli selected at the calli stage. However, there were no significant differences in phenotypic abnormality percentages among plantlet populations regenerated from non-transformed (P1), transformed non-selected (P2), and transformed calli selected at the embryogenic stage (P4). Our research findings clearly imply that stress caused by injuries (by particle bombardment) and subsequent selection has no significant effect on phenotype of regenerated plantlets of oil palm E. guineensis Jacq. Tenera. Key words: Calli, oil palm, plantlet regeneration, somaclonal variation INTRODUCTION Oil palm Elaeis guineensis Jacq. Tenera (EgT) is cultivated in the tropics for its oil, which is used widely in the food industry and has the potential to substitute diesel as a fuel (Moretzsohn et al. 2000). Currently, oil palm is the second major source of vegetable oil in the world vegetable oil market (World production of 17 oils and fats: 1994–2001), and it is predicted to supply 50% of the market by 2020 (Rajanaidu and Jalani, 1995). Malaysia is the largest producer of palm oil in the world, but cultivation is expanding, especially in countries of South-East Asia and Africa. In several laboratories worldwide research is going on to improve oil palm by genetic manipulation. However, 5% to 80% of the oil palm plantlets regenerated through tissue culture methods are known to generate abnormalities in their floral development, involving an apparent feminisation of male parts in flowers, which is *Corresponding author; e-mail: [email protected]


called “mantled” phenotype (Corley et al. 1986, Jaligot et al. 2002). Rival et al. (1998b) has reported the 100% reversion of the slightly “mantled” individuals, and 50 % reversion of the severely “mantled” ones, but only after 9 years in the field. The random amplified polymorphic DNA (RAPD) technique has been employed for the analysis of genetic variation (Shah et al. 1994), but was unsuccessful in the detection of somaclonal variants among regenerated populations (Rival et al. 1998a). In addition to this, several potential markers of the oil palm ‘mantled’ abnormality, such as polypeptide patterns (Marmey et al. 1991) and endogenous cytokinins (Besse et al. 1992) have been investigated, but difficulties have been encountered when using these procedures on a large scale. The induction and expression of the abnormalities in oil palm plantlets has been linked to the 2, 4-dichlorophenoxy acetic-acid (2,4-D) (Bayliss 1973, Sogeke 1998), and cytokinins by some reports in last decade (Agamuthu and Ho 1992, Besse et al. 1992, Duval et al. 1995, Ho et al. 1991). 613

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In our study of the stearoyl-acyl-carrier protein (ACP) desaturase gene (GenBank Accession No: AF507965) (Shah et al. 2000) silencing by the antisense construct, we monitored, and recorded the time required for regeneration of plantlets from non-transformed, and transformed calli of EgT through somatic embryogenesis, in the absence of 2,4-D and cytokinins. Moreover, we analyzed the plantlets both qualitatively and quantitatively based on visible phenotypic variation in four different populations of regenerated plantlets before hardening, and this is reported here. The objective of the study was to find out the effect of stress caused by particle bombardment induced injuries in target tissue and subsequent selection pressure on the phenotype of plantlets, regenerated through somatic embryogenesis from non-transformed and transformed calli of EgT. MATERIALS AND METHODS Immature zygotic embryos (IZEs), which were used as explants in this study were isolated (aseptically) from twelve-week-old [Weeks After Anthesis (WAA)] fruits of EgT, procured from the Pamol Research Station, Kluang, Johor, Malaysia. Transformation vector Transformation vector pADST35 (Fig. 1), which was constructed earlier in our lab with partial (1161 bp) antisense sequence of stearoyl-ACP desaturase gene from pTD7 cDNA clone (GenBank Accession No. U68756) (Shah et. al. 2000) was used in this study. The backbone of pADST35 is derived from pAHC25 (Christiensen et al. 1992). In pADST35 both the stearoyl-ACP desaturase gene and the bar gene [resistance to phosphinotricin (PPT) as a selection marker] were driven by the maize ubiqutine promoter.

P stI U biqutine

B glII

Intron

Callus induction and maintenance Callus was induced from 800 IZEs, which were removed aseptically in a laminar hood and were inoculated in petri plates (90 × 15 mm) containing modified MS (Murashige and Skoog 1962) basal semisolid medium. Medium (MS) supplemented with 2 mg l -1 a-Naphthaleneacetic acid (NAA), 100 mg l -1 casein hydrolysate and 30 g l-1 sucrose was designated as CMSII medium. By subculturing at 6-8 weeks interval, the induced calli were maintained on similar medium up to 8 months at 27°C (±2) in the dark. Transformation, selection and somatic embryogenesis of calli Eight-month-old calli were used as target tissue for the transformation with antisense stearoyl-ACP desaturase. Transformation vector pADST35 DNA was delivered in the target tissue (calli) by employing the combination of all optimized physical parameters reported by Parveez et al. (1997) with minor modifications. For comparison one group of calli, which were not bombarded at all for gene transfer, were maintained as control. Three days after transformation, putatively transformed calli were divided into three groups. The first group of calli was maintained on nonselection medium (CMSII only). The second group of calli was transferred on to selection medium [CMSII + 8 mg l-1 PPT] and the third group of calli was maintained on non-selection medium (CMSII only) initially, which then underwent selection stress just after the embryogenic stage of the calli. All four groups of calli viz., control, transformed not-selected, transformed selected at calli stage, and transformed selected at embryogenic stage were maintained on CMSII medium in the dark until embryogenesis. Somatic embryos from each group were moved from CMSII medium onto

BamH I A ntisense stearoyl-A C P desaturase

Fig. 1. Schematic diagram of the antisense desaturase gene cassette. The arrow passing through ubiqutine promoter box, antisense stearoyl-ACP desaturase gene box, and NOS terminator box indicates the direction of transcription (Details of the transformation vector pADST35 will be available upon request).

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Table 1. Categories of the plantlets examined. P stands for population Category

Plantlets regenerated from

P1

Non-transformed calli (Control) Transformed non-selected calli Transformed calli, selected at calli stage Transformed calli, selected at embryogenic stage of calli

P2 P3 P4

Number*

60 50 42 50

*Total number of plantlets regenerated, and examined for phenotypic variation.

The percentage of the phenotypic abnormality was calculated for each population separately. Mean shoot and root lengths were calculated separately for the normal and abnormal plantlets in all four populations. The Duncan’s Multiple Range Test (DMRT), available in Statistical Analysis System (SAS) program (Edition 6.12) was used for the statistical analysis of the data. RESULTS AND DISCUSSION Calli were successfully induced from 800 IZEs. In eight months enough calli were regenerated from the induced calli for the transformation. Calli, at the time of particle bombardment, were fragile and pale yellowish in color. Transformation of 8-month-old calli with pADST35 transformation vector was successfully completed in aseptic conditions by employing the combination of all optimized physical parameters. Selection of transformed calli, which underwent Indian J. Plant Physiol., 2003 (Special Issue) pp. 613-619

selection stress immediately after particle bombardment, and another group of transformed calli (selected at embryogenic stage of calli) were successfully selected using an optimized concentration of PPT (8 mg l-1 PPT). Non-transformed calli (Control), transformed nonselected calli, transformed calli which underwent selection stress immediately after particle bombardment, and another group of transformed non-selected calli (selected at embryogenic stage of calli) entered into the embryogenic state after 4, 9, 13, and 9 months, respectively after particle bombardment. To complete caulogenesis and rhizogenesis, somatic embryos from all four groups were in culture for different periods. Fig. 2 shows the period of in vitro incubation for all four groups of plantlets. Total Incubation Time (months)

semisolid Y3 medium (Rillo and Paloma 1990), which was supplemented with 1 mg l-1 NAA, and 30 g l -1 sucrose for maintenance until caulogenesis was over. During caulogenesis somatic embryos were exposed to 14 hour photoperiod (photon flux 150 µmol m-2 s-1 at 28°C). After caulogenesis, cultures were moved onto rooting medium [MS macro and micro nutrients + Y3 vitamins + 0.1 mg l -1 NAA, 0.65% phytagar ® (commercial Grade- GIBCOBRL), and pH was adjusted to 5.8]. Rooted plantlets were taken out from the test tubes for hardening treatment. At this juncture, all plantlets were examined for phenotypic variation in all four populations (Table 1).

40

a

35

b

30 25

c

c

P1

P2

20 15 10 5 0

P3

P4

Fig. 2. Total in vitro incubation time (in months) of P1, P2, P3, and P4. Definitions of P1-P4 are given in Table 1. Bars indicate the standard deviation. Means with the different letters are significantly different at P=0.05.

Phenotypic variation was observed in all four groups studied, P1-P4. Plantlets from all 4 groups were grouped into two categories, normal plantlets, and plantlets showing grassy shoot appearance and stunted growth were designated as abnormal plantlets. The leaves of normal plantlets were 2 times broader compared to leaves of abnormal plantlets, and the leaves of the normal plantlets were dark green in color (Fig. 3a) in comparison to leaves of the abnormal plantlets (Fig. 3b). Interestingly abnormal plantlets rooting was profuse (Fig. 3c), and roots were thick, slightly greenish, and with blunt ends in comparison to the roots of normal plantlets (Fig. 3d). In addition to this, it was noted that 615

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Fig. 3a

Fig. 3b

Fig. 3d

Fig. 3c

Fig .3e g. Fig. 3(a) Leaves of normal plantlet, (b) leaves of abnormal plantlet, (c) rooting pattern of abnormal plantlet, (d) rooting pattern of normal plantlet, (e) abnormal leaves from normal looking plantlets.

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among normal plantlets of all four groups, a few (< 3%) plantlets had 1 or 2 abnormal leaves (Fig. 3e). This disorder/abnormality of leaves reverted completely in 2 to 4 months during hardening of the plantlets.

plantlets was 21.2 cm (± 4.3), while it was 8.9 cm (± 0.9) for abnormal plantlets. Whereas mean root length was 7.7 cm (±3.5), and 5.3 cm (±2.6) for normal, and abnormal plantlets respectively (Fig. 4b).

The display of phenotypic abnormalities in P3 was the highest (40.4%), while it was 33.3% in P1, 32.0% in P2, and 32% in P4 (Fig. 4a). There was no significant difference in the mean shoot, and root lengths within the four normal and abnormal groups of plantlets when plantlets were moved from in vitro to in vivo for the hardening treatment. The mean shoot length for normal

The collective action of both genetic and environmental factors is known to influence the phenotype of organisms. Methylation of deoxycytidine (dC) residues was shown to be involved in the regulation of genes at the transcriptional level, particularly during the differentiation/dedifferentiation process in addition to environmental factors (Finnegan et al. 1993, 1998, Phillip et al. 1994). This hypothesis was reconfirmed by findings reported by Jaligot et al. (2000). It has been reported that the use of cytokinins in the medium induces the abnormalities, which may not appear until years later (Agamuthu and Ho 1992, Besse et al. 1992).

80

a

70

Abnormal

Normal

a

a

a b

% Plantlets

60 50 40

c

d

d

d

30 20 10 0

b

30

Shoot/root length (cm)

P1

25

P2

P4

a Normal Abnormal

20 15

P3

b a

10

b

5 0 Shoot

Root

Fig. 4(a) Phenotype in four different populations of the oil palm EgT plantlets regenerated through somatic embryogenesis. (b) Mean length of shoots and roots (in cm) of normal and abnormal plantlets of EgT. Bars indicate the standard deviation. Means with the different letters are significantly different at P=0.05. Duncans Multiple Range Test (DMRT) was carried out separately for statistical analysis of mean lengths of shoots and roots of normal and abnormal plantlets (Fig. 4b).


In our experiments, we did not use any cytokinins. Therefore, if the DNA methylation caused by cytokinins is the main cause of phenotypic abnormality in oil palm then one interpretation of the observed phenotypic abnormalities in our experiments is that the plantlets showing phenotypic abnormality might have high level of endogenous cytokinin. The 2,4-D is known to cause mitotic spindle abnormality (Bayliss 1973), which is associated with induction of the abnormalities in oil palm (Sogeke 1998). In order to avoid/minimize the number of somaclonal variants we used NAA for the induction of calli from IZEs. The occurrence of phenotypic abnormalities in the plantlets regenerated in our experiments in the absence of 2,4-D, and cytokinins, argues that the NAA might be playing a role in the induction of phenotypic abnormalities. The percentage of the phenotypic abnormalities in P3 (40.4%) is 7.1% higher when compared to the control (P1), and 8.4% higher in comparison to both P2, and P4. However, the in vitro incubation time was the longest for P3. It was in vitro for 16, 11, and 6 months extra time in comparison to P1, P2, and P4 respectively. There were no significant differences in the abnormality percentage among P1, P2, and P4. However, high (7.1 %) percentage of the phenotypic abnormality in P3 could not be attributed to the prolonged in vitro incubation, as the difference is minor relative to the overall levels of phenotypic variation, eventhough phenotypic 617

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abnormality is attributed to the prolonged in vitro incubation by Rohani et al. (2001). The non-significant phenotypic abnormality percentage among P1, P2 and P4 clearly implies that the stress caused by particle bombardment (injuries) and selection has no significant impact on phenotype of regenerated oil palm plantlets. In addition, the occurrence of phenotypic variation in the absence of 2,4D and cytokinins suggests the involvement of multiple genetic and epigenetic causal mechanisms in phenotypic abnormality induction, which is in agreement with the hypothesis made by Kaeppler et al. (2000). Recently, Jaligot et al. (2002) has proposed that methylationsensitive RFLPs could be used for the early detection of somaclonal variation during early stages of clonal propagation. However, to examine the cause of these reported phenotypic abnormalities more closely in oil palm EgT more research is needed. ACKNOWLEDGEMENTS The authors are grateful to the Ministry of Science and Technology of Malaysian Government for funding (Grant No IRPA: 09-02-02-0161), to the Pamol Research Station, Kluang, Johor, Malaysia for supplying fruits of Elaeis guineensis Jacq. Tenera for this study, and to Mr. Raai for his help in photography.

Duval, Y., Besse, I., Verdeil, J.L. and Maldiney, R. (1995). Study on the induction of the floral morphogenesis abnormality in oil palm during the in vitro regeneration process. In: V. Rao, I.E. Henson and N. Rajanaidu (eds.), Recent Developments in Oil palm Tissue Culture and Biotechnology. Palm Oil Research Institute of Malaysia (PORIM), Kuala Lumpur, pp. 64-69 (Proceedings of the 1993 ISOPB International Symposium on Recent Developments in Oil Palm Tissue Culture and Biotechnology, Kuala Lumpur, 24-25 September, 1993). Finnegan, E.J., Brettell, R.I.S. and Dennis, E.S. (1993). The role of DNA methylation in the regulation of plant gene expression. In: J.P. Jost and H.P. Saluz (eds.), DNA Methylation: Molecular Biology and Biological Significance, pp. 218-261. Birkhauser, Basel. Finnegan, E.J., Genger, R.K., Peacock, W.J. and Dennis, E.S. (1998). DNA methylation in plants. Annu. Rev. Plant Mol. Biol. 49: 223247. Ho, C.C., Lim, A.L., Agamuthu, P., Nizar, A., Ramien, A. and Thievendirajah, K. (1991). The cellular basis and possible cause for the development of mantled flower in oil palm derived from tissue culture. National Seminar IRPA (Strategic Sector) 16-19 Dec., 1991, Penana, Malaysia. Jaligot, E., Beule, T. and Rival, A. (2002). Methylation-sensitive RFLPs: characterization of two oil palm markers showing somaclonal variation-associated polymorphism. Theor. Appl. Genet. 104: 1263-1269. Jaligot, E., Rival, A., Beule, T., Dussert, S. and Verdeil, J.L. (2000). Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis. Plant Cell Rep. 19: 684-690.

REFERENCES

Kaeppler, S.M., Kaeppler, H.F. and Rhee, Y. (2000). Epigenetic aspects of somaclonal variation in plants. Plant Mol. Biol. 43: 179-188.

Agamuthu, P. and Hom, C.C. (1992). Quantification of endogenous cytokinins in the flower primordial of normal seed-derived and mantled tissue culture-derived oil palm. Trans Malaysian Soc. Plant Physiol. 3: 143-150.

Marmey, P., Besse, I. and Verdeil, J.L. (1991). Mise en evidence d’um marqueur proteique differenciant deux types de cals issus du meme clone chez le palmier a huile (Elaeis guineensis Jacq). CR Acad. Sci. Paris (III) 313: 333-338.

Bayliss, M.W. (1973). Origin of chromosome number variation in cultured plant cells. Nature 246: 529-530.

Moretzsohn, M.C., Nunes, C.D.M., Ferreira, M.E. and Grattapaglia, D. (2000). RAPD linkage mapping of the shell thickness locus in oil palm (Elaeis guineensis Jacq.). Theor. Appl. Genet. 100: 6370.

Besse, I., Verdeil, J.L., Duval, Y., Sotta, B., Maldiney, R. and Miginiac, E. (1992). Oil palm (Elaeis guineensis Jacq.) clonal fidelity: endogenous cytokinins and indolacetic acid in embryogenic callus cultures. J. Exp. Bot. 43: 983-989. Christiensen, A.H., Sharrock, R.A. and Quail, P.H. (1992). Maize polyubiquitine gene: thermal perturbation of expression and transcript splicing and promoter activity following transfer to protoplast by electroporation. Plant Mol. Bio. 18: 675-689. Corley, R.H.V., Lee, C.H., Law, L.H. and Wong, CY. (1986). Abnormal flower development in oil palm clones. Planter (Kuala Lumpur) 62: 233-240.

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Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. Parveez, G.K.A., Chowdhury, M.K.U. and Saleh Norihan, M. (1997). Physical parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) using the biolistic device. Industral. Crops Prod. 6: 41-50. Phillip, R.L., Kaeppler, S.M. and Olhoft, P. (1994). Genetic instability of plant tissue cultures: breakdown of normal controls. Proc Natl. Acad. Sci. 91: 5222-5226. Indian J. Plant Physiol., 2003 (Special Issue) pp. 613-619

PHENOTYPIC VARIATION IN OIL PALM Rajanaidu, N. and Jalani, B.S. (1995). World-wide performance of DxP planting materials and future prospects. In: Proceedings of the 1995 PORIM National Oil Palm Conference. Technology in Plantation “The Way Forward”. Kuala Lumpur, Malaysia, pp. 1-29. Rillo, E.P. and Paloma, M.B.F. (1990). Comparison of three media formulations for in vitro culture of coconut embryos. Oleagineux. 45: 319-323. Rival, A., Bertrand, L., Beule, T., Combes, M.C., Trouslot, P. and Lashermes, P. (1998a). Suitability of RAPD analysis for the detection of somaclonal variants in oil palm (Elaeis guineensis Jacq.). Plant Breed. 117: 73-76. Rival, A., Treger, J., Verdeil, J.L., Richaud, F., Beule, T., Hartman, C., Rode, A. and Duval, Y. (1998b). Molecular search for mRNA and genomic markers of the oil palm “mantled” somaclonal variation. Acta Hort. 461: 165-171.


Rohani, O., Sharifah, S.A., Mohd. Rafii, Y., Ong, M., Tarmizi, A.H. and Zamzuri, I. (2001). Tissue culture of oil palm. In: B. Yusof, B.S. Jalani and K.W. Chan (eds.), Advances in Oil Palm Research, Malaysian Palm Oil Board, Malaysia. Shah, F.H., Rashid, O., Simons, A.J. and Dunsdon, A. (1994). The utility of RAPD markers for the detection of genetic variation in oil palm (Elaeis guineensis). Theor. Appl. Genet. 89: 713-718. Shah, F.H., Rashid, O. and Cha, T.S. (2000). Temporal regulation of two isoforms of cDNA clones encoding delta-9-stearoyl-ACP desaturase from oil palm (Elaeis guineensis). Plant Sci. 152: 2733. Sogeke, A.K. (1998). Stages in the vegetative propagation of oil palm, Elaeis guineensis Jacq. through tissue culture. J. Oil Palm Res. 10: 1-9. World production of 17 oils and fats. (1994–2001). http:// 161.142.157.2/home2/home/ ei_table6_3.htm

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