Introduction

Mass propagation of elite oil palm material through tissue culture has been used for a number of years in several laboratories (Abdullah et al. 2005). Sometimes, large scale in vitro clonal propagation of oil palm is hindered by the occurrence of truncated leaf syndrome (TLS), a somaclonal abnormality (Tan et al. 1996). truncated leaf syndrome is a vegetative abnormality that is only prevalent upon transplantation to the nursery. The affected seedlings bear leaves which are truncated at the tip and in severe cases it remains as an irreversible stub (Tan et al. 1999). Our recent phenotypic and morpho-histological investigations on truncated leaf syndrome seedlings showed that these plants were of less vigor than wild type with reduced plant height, pale green leaves, decreased number of roots, smaller shoot apical meristem and drastically deformed stomata (Habib et al. 2012). Depending on the frequency of truncated leaf syndrome occurrence in a clone, the truncated leaf syndrome seedlings were categorized into severe, moderate and mild. We also reported that the severity of truncated leaf syndrome increased with the reduction of shoot apical meristem size. Several altered anatomical features such as depressed epidermal layer, longer epidermal and bigger sub-epidermal cells with unorganized inter cellular spaces were also observed in truncated leaf syndrome seedlings. Other aberrations include impaired root tip with reduced lateral roots and/or undifferentiated vascular systems. The cause of truncated leaf syndrome occurrence could be due to many factors including environmental, nutritional deficiency, hormonal imbalance, genetic changes in the somatic cell during micro propagation, or epigenetic causes. Truncated leaf syndrome seedlings with mild symptoms recovered when placed in shade suggesting that it could be an environmental factor (Tan et al. 1996). Tan et al. (1999) also documented that the mildly affected truncated leaf syndrome seedlings recovered upon treatment with boron fertilizers, implying boron deficiency could be a cause of this abnormality. To date, there is no detailed information on the endogenous hormone and mineral levels of the truncated leaf syndrome seedlings. Therefore, this study was undertaken to determine the chlorophyll, mineral, cytokinin and brassinosteroid levels in truncated leaf syndrome seedlings in comparison to the wild type of tissue culture-derived oil palm seedlings.

Materials and methods

Frozen oil palm (Elaeis guineensis Jacq. var. tenera) leaf samples from nine clones (wild type and truncated leaf syndrome seedlings from each clone of the same age and parentage) were used for biochemical, cytokinin and brassinosteroid analyses.

Approximately, 0.2 g of leaf samples were lyophilized, ground, and extracted using the dry digestion method according to Jones (1991) and Gupta (1993). The levels of boron, zinc, calcium, magnesium and potassium were determined using the ICP-OES (Perkin Elmer Optima 2000DV, Connecticut, USA). Chlorophyll was extracted from truncated leaf syndrome and wild type leaf samples (0.2 g) and determined according to the method of Moran and Porath (1980). The concentration of total chlorophyll, Chl a and Chl b were calculated according to Arnon (1949). The quantification of different cytokinin derivatives was carried out according to the method described by Novák et al. (2008). Approximately 50 mg of each sample were extracted with 1,000 μl of Bieleski buffer containing 60 % methanol, 25 % CHCl3, 10 % H2O and 5 % HCOOH. Internal standard mixtures (1 pmol of each compound per sample) of [13C5]tZ, [2H5]tZR, [2H5]tZ9G, [2H5]tZOG, [2H5]tZROG, [2H5]tZRMP, [13C5]cZ, [2H3]DHZ, [2H3]DHZR, [2H3]DHZ9G, [2H7]DHZOG, [2H3]DHZRMP, [2H6]iP, [2H6]iPR, [2H6]iP9G, [2H6]iPRMP, [2H7]BA, [2H7]BAR, [2H7]BA9G, [2H7]BARMP, [15N4]mT, and [15N4]oT were added to the sample. The extracts were purified through two ion-exchange chromatography steps (SCX, DEAE-Sephadex combined with SPE C18-cartridges) which yielded two fractions, the first containing cytokinin free bases, ribosides, 9-glucosides and O-glucosides, the second fraction contained ribonucleotides. Immunoaffinity purification was conducted according to Novák et al. (2003). The samples were then analyzed by ultra-performance liquid chromatography-electro spray ionization mass spectrometry [UPLC–ESI(+)–MS/MS]. Determination of brassinosteroids in the leaf was performed using the method of Swaczynova et al. (2007) with minor modifications. Approximately 50 mg each sample was extracted in ice–cold 80 % methanol with 50 pmol of [2H6]brassinolide and [2H6]castasterone as internal standards. Then the samples were purified on Strata X polymeric reversed-phase columns (500 mg, 33 μm, Phenomenex) followed by purification using HLB polymeric RP-cartridges (200 mg, 30 μm, Waters, Milford, MA, USA). The eluant was then evaporated to dryness in a Speed-Vac (UniEquip), reconstructed in solution corresponding to the mobile phase at the initial conditions of HPLC run and analyzed with a liquid chromatography–mass spectrometry system (Quattro micro API, Waters, USA). Tandem mass spectrometry data was then analyzed using the software MassLynx 4.1 (Waters, USA) and quantified by the standard isotope-dilution method. All data obtained were presented as means of standard errors and the difference between the truncated leaf syndrome and wild type and within the truncated leaf syndrome groups were computed by paired t test accessible from the internet (http://www.quantitativeskills.com/sisa/statistics/t-test.htm) with P < 0.01. The samples of about 5 mg in weight were analyzed for IAA content as described by Pencik et al. (2009).

Results and discussion

Biochemical analysis showed that the boron level was significantly higher in the wild type leaves which were almost double than that of truncated leaf syndrome groups (Fig. 1), indicating the truncated leaf syndrome phenotype could be as a result of low boron condition. This finding was consistent with the observations by Tan et al. (1999) who stated that the truncated leaf syndrome abnormality was caused by boron deficiency, as mildly affected truncated leaf syndrome seedlings recovered after boron application. However, the results of our study also showed that the level of boron was not significantly different among the three different truncated leaf syndrome groups suggesting that boron deficiency was not the main cause of truncated leaf syndrome. Perhaps there are other factors that trigger the truncated leaf syndrome abnormality. The anatomical alterations observed in truncated leaf syndrome seedlings such as hypertrophy, decreased number of vascular bundles, poorly developed vascular system and impairment of root tip (Habib et al. 2012) could be caused by the low boron level in truncated leaf syndrome seedlings. Boron is an essential micronutrient for normal growth and development of vascular plants (Maziah et al. 2009). Under boron deficient conditions, hypertrophy and less developed vascular bundles were observed in carrot (Warington 2008) and a small number of vascular bundles were also observed in cotton plants (Oliveira et al. 2003). The truncated leaf syndrome seedlings possessed smaller shoot apical meristem than those of the wild type, possibly caused by the lower boron level. This finding is similar to the results reported by Tanaka and Fujiwara (2008) whereby the growth of both root and shoot meristems of squash and sunflowers were inhibited under boron deficient condition. In addition, zinc level was also found to be significantly lower in severe and moderate truncated leaf syndrome seedlings than those of wild type (Fig. 1). This could be attributed by the lower boron levels which can influence either uptake or transport of mineral elements in plants. Pilipenko and Solv’eva (1979) reported that zinc uptake was decreased in boron-deficient navy bean plants. Zinc is one of the essential micronutrients with a significant role in many vital metabolic processes (Aravind and Prasad 2005).

Fig. 1
figure 1

Mineral composition of truncated leaf syndrome leaves in comparison to wild type leaves (B Boron, Zn Zinc, Mg Magnesium, Ca Calcium and K Potassium). a B, Zn, Mg, Ca and K content in the severe truncated leaf syndrome group compared to wild type seedlings; b moderate truncated leaf syndrome group; and c mild truncated leaf syndrome group. B and Zn concentration were determined in mg kg−1 dry weight and Mg, Ca and K concentration were determined in g kg−1 dry weight. Each value represents the mean of three clones (four replicates in each clone). Same letters are not significantly different. The data is significant at 5 % (P < 0.05)

Full size image

Our study showed that the chlorophyll contents in all three categories of truncated leaf syndrome were significantly lower compared to wild type (Fig. 2). Hence, the leaf appeared paler green in truncated leaf syndrome seedlings than that of the control. Moreover, the lower chlorophyll content could be due to lower zinc in truncated leaf syndrome seedlings. The growth of the plant, especially shoot growth was severely suppressed and chlorophyll concentration was decreased under low zinc status in bean (Cakmak et al. 1989). Therefore, the present findings of this study imply that the severe phenotype of truncated leaf syndrome seedlings might be associated to both zinc and boron deficiency.

Fig. 2
figure 2

Chlorophyll content (mg/g FW) of truncated leaf syndrome leaves in comparison to wild type leaves. a Chlorophyll content of severe truncated leaf syndrome group compared to wild type seedlings; b moderate truncated leaf syndrome group; c mild truncated leaf syndrome group and d chlorophyll content in the three truncated leaf syndrome groups. Each value represents the mean of three clones (four replicates in each clone). Same letters are not significantly different. The data is significant at 5 % (P < 0.05)

Full size image

Among the phytohormones, cytokinins have proven to be the most important factor for histological changes (Tabori-Magyar et al. 2010). In addition, cytokinins can also directly affect mineral uptake and distribution patterns (Mengel and Kirkby 2001) which can lead to the formation of abnormal plantlets in tissue culture. The present results revealed that some of the cytokinin derivatives such as tZ: trans-zeatin; tZR: trans-zeatin riboside; tZOG: trans-zeatin O-glucoside; tZR5′MP: trans-zeatin riboside 5′monophosphate and cZR5′MP: cis-zeatin riboside 5′monophosphate were significantly higher in severe truncated leaf syndrome plants than that of wild type (Fig. 3). The higher level of cytokinins in this study might be due to the overproduction and accumulation of cytokinins which could have caused severe abnormality in the TLS. Increased cytokinin level in the culture medium can affect the mineral composition of explants which was associated with abnormal shoot morphology (Ramage and Williams 2004). Hence, the higher cytokinin levels perhaps adversely affected the uptake of boron and zinc in the severe truncated leaf syndrome seedlings. We have also carried out some preliminary measurement of free IAA and the results showed no significant differences between the WT and TLS (data not shown).

Fig. 3
figure 3

Cytokinins levels (pmol/g dry weight) in severe truncated leaf syndrome leaves in comparison to wild type. tZ trans-zeatin, tZR trans-zeatin riboside, tZOG trans-zeatin O-glucoside, tZROG trans-zeatin riboside O-glucoside, tZ9G trans-zeatin 9-glucosides, tZR5′MP trans-zeatin riboside 5′mono phosphate, cZ cis-zeatin, cZR cis-zeatin riboside, cZOG cis-zeatin O-glucoside, cZROG cis-zeatin riboside O-glucoside, cZ9G cis-zeatin 9-glucosides, cZR5′MP cis-zeatin riboside 5′mono phosphate. Each value represents the mean of three clones (two replicates in each clone). Same letters are not significantly different. The data is significant at 5 % (P < 0.05)

Full size image

In severe truncated leaf syndrome leaves, brassinolide (BRs) levels were significantly higher compared to that of the wild type (Fig. 4). The general effects of brassinosteroids are in the promotion of cell elongation, cell division, differentiation, disease resistance, stress tolerance and senescence throughout a plant’s life cycle (Mussig 2005). The decreased numbers of leaf mesophyll and root cortex parenchyma cells in truncated leaf syndrome were perhaps due to the inhibitory effect of brassinosteroids on cell division. Impaired vascular bundles with undifferentiated phloem in severely affected truncated leaf syndrome leaves (Habib et al. 2012) could be attributed to highly superoptimal brassinosteroids levels. This result was consistent with the studies of Nakamura et al. (2006) who reported that BRs inhibit the differentiation of phloem. To maintain homeostatic levels, active brassinosteroids in plants must be deactivated or degraded after exerting their activity (Kim et al. 2005). Therefore, the severity in the truncated leaf syndrome phenotype might be due to the high accumulation of brassinolide too. Thus, the present study implies that excessive synthesis and accumulation of cytokinins and brassinosteroids might affect the boron and zinc uptake directly or indirectly.

Fig. 4
figure 4

Brassinosteroid (brassinolide) levels (pmol/g dry weight) in severe truncated leaf syndrome leaves in comparison to wild type. Each value represents the mean of three clones. Error bars represent the SE of means. The data is significant at 5 % (P < 0.05)

Full size image