Abstract: In the present study, various explants of Murraya paniculata (Jack) Linn., such as cotyledons, shoots and young stems were cultured on MS medium supplemented with various concentrations of Benzyl Amino Purine (BAP) under 25 ± 1 °C with 16 h light and 8 h dark and also 8 h light and 16 h dark to obtain complete plant regeneration. In vitro flowering was observed from shoot explants cultured on MS supplemented with 0.5-2.0 mg L-1 Naphthalene Acetic Acid (NAA) and also on MS basal medium under similar conditions. The leaves and flowers obtained from both in vivo and in vitro conditions were examined and compared. Morphological studies such as leaf clearing, epidermal peeling were studied using light and scanning electron microscope. Macromorphological studies of the flowers produced from in vivo and in vitro conditions were also examined. Morphologically, there were no differences between in vivo and in vitro flowers except the flowers produced from tissue culture systems were smaller in size with protruding stigmas. Differences were also found in the number of layers of palisade cells and the presence or absence of epicuticle layer of the leaves. Leaves produced from tissue culture system were smaller in size with membranous texture. Stomata were present only on the abaxial surfaces of both in vivo and in vitro leaves but the stomata were raised above the epidermis in the latter.
INTRODUCTION
Tissue culture is done for various purposes, for instance; for mass propagation, haploid and triploid plant production, somatic hybridisation, genetic improvement, production of secondary metabolites, production of synthetic seeds and also for fundamental understanding of plant basic processes such as division, differentiation and development (Bhojwani and Razdan, 1996). Comparison of processes that occur in cells under in vitro environment with that of intact plants can give some understanding of the underlying mechanisms involve. Cells grown in vitro are much easier to manipulate compared with the cells in intact plant. Plants derived from tissue culture systems can result in abnormalities or variations either at cellular or morphological level (Larkin and Scowcroft, 1981). Some changes that occur at cellular level is expressed morphologically, whilst some plants such as Vicia faba can tolerate the cellular variations and nothing is revealed at morphological level (Taha and Francis, 1990).
In this study, we would like to report some tissue culture studies of Murraya paniculata (Jack) Linn. until floral stage in tissue culture system. The leaves and flowers produced in vitro were then compared with those produced in vivo. The main objective being to investigate whether there are variations between the leaves and flowers obtain under different conditions. For cloning purposes, it is desirable to obtain plants which are the exact copies of the parents, however, variations may be beneficial in certain cases.
MATERIALS AND METHODS
Seeds of Murraya paniculata obtained from Rimba Ilmu, University of Malaya, Kuala Lumpur, were washed and sterlised in the usual manner (Taha, 1993) with 70% (v/v) alcohol, 2.5% (w/v) sodium hypochlorite and 0.1% (v/v) Tween-twenty. The seeds were then cultured on MS basal medium (Murashige and Skoog, 1962) to get explant sources. The seedlings used to initiate cultures were 3-4 weeks old. Various explant organs such as leaves, cotyledons, stems and shoots were cut into small pieces (5 mm for shoot and stem and 5x5 mm for leaf and cotyledon). These explants were then cultured on MS media supplemented with different concentrations of NAA and BA. At least 20 replicates were used for each treatment and each treatment was repeated 3 times. The cultures were maintained under 16 h light and 8 h darkness and also 8 h light and 16 h darkness at 25±1°C. The cultures were examined every alternate day. The leaves and flowers obtained from in vitro systems were then compared with those of intact plants.
Standard procedures were followed for SEM and two to four specimens were examined. A section of 5 mm2 of the dry leaf lamina (both adaxial and abaxial surfaces) was fixed on to double sided adhesive tape on labelled stubs. The specimens were coated with gold using a Bio-Rad SEM coating system and scanned in Philips SEM 515 Scanning electron microscope. For epidermal studies, samples were taken from midway between the base and apex of lamina. The epidermal characters were investigated from cuticular membrane preparations. Each sample was macerated in 10% nitric acid until the epidermis could be removed. Adaxial and abaxial epidermis were then separated using fine forceps and a dissecting needle. The cleaned epidermis were stained in safranin and mounted in Canada balsam. The method used for anatomical study was after Johansen (1940) with minor modifications. Sectioning of the midrib of the leaf lamina was done using a Reichart Rotary microtome. Photographs were taken using a Photomicroscope. Data obtained are presented in Tables 2 and 3. Light micrographs are shown in Figs. 1, 2, 9 and 10. Scanning electron micrographs of both adaxial and abaxial leaf surfaces are shown in Figs. 3-8. The terminology used is that of Dilcher (1974).
RESULTS
Complete plant regeneration of Murraya paniculata was achieved from portions of cotyledons, shoots and young stems cultured on MS supplemented with 1.0 mg L-1 BA and also MS basal medium. Rooting of this species could be obtained on either MS basal or MS containing 1.0 mg L-1 NAA. In vitro flowering was obtained from MS basal and also on MS added with 0.5-2.0 mg L-1 NAA. In vitro flowering could only occur when cotyledons and shoots were used, but not from plantlets derived from stem and leaf explants. Callus was routinely observed from leaf and stem explants. In vitro flowering occurred after 26 days on MS basal medium under 16 h light and 8 h dark but it took longer to flower (35 days) under 8 h light and 16 h darkness on the same medium. The percentage of flowering reduced from 80 to 60% when the cultures were exposed to shorter day length. By adding 0.5-2.0 mg L-1 NAA, the in vitro flowering was found to occur faster (21 days) (Table 1). GA3 was not effective for in vitro flowering of this species although it may be effective for other species. The leaves
| Table 1: | The percentage flowering of Murraya paniculata cultured on MS medium supplemented with different concentrations of NAA and BA (mg L-1) at 25±10°C |
| Table 2: | Vegetative and reproductive morphological characters of Murraya paniculata plant in vivo and in vitro |
| Table 3: | Micromorphological and anatomical characters of the leaf lamina of Murraya paniculata in vivo and in vitro |
and flowers obtained from the regenerants were then subjected to micromorphological and anatomical studies (Tables 2, 3).
Morphologically, there were no differences in the vegetative and reproductive characters between in vivo and in vitro plant of Murraya paniculata except for the sizes and textures of the leaves, the sizes of the flowers and the type of stigmas produced. The size of in vitro
| Fig. 1: | Light micrograph of adaxial surface of leaf showing the anticlinal wall of the epidermis (AC) |
| Fig. 2: | Light micrograph of abaxial surface of leaf showing anomocytic stomata and anticlinal wall of the epidermis (AC) |
leaves ranges from 1.5-2.0x0.5-1.0 cm whereas in vivo leaves were larger in size, ranging from 3.0-5.0x1.5x2.0 cm. The texture of in vitro leaves was membranous while in vivo leaves were coriaceous. Trichomes were observed on the abaxial surfaces of both types of leaves but in vitro leaves produced a smooth type of trichome (Figs. 6, 8). Palisade cells were uniseriate in in vitro species while they were biseriate in in vivo species (Figs. 9, 10). In vitro flowers were smaller in size compared with in vivo flowers and the former also produced protruding stigmas. Studies on micromorphological and anatomical characters of the leaf lamina of in vivo and in vitro species of Murraya paniculata also showed little differences between in vitro and in vivo leaves. Both leaves were dorsiventral and hypostomatic. Stomata on in vitro leaves were observed to be raised above the epidermis but in in vivo leaves the stomata were found to
| Fig. 3: | Scanning electron micrograph of abaxial surface of in vivo leaf showing the distribution of stomata |
| Fig. 4: | Scanning electron micrograph of abaxial surface of in vivo leaf showing the structure of the trichome |
be leveled with the epidermis. The stomata were dispersed randomly over the whole abaxial surface and they appear rounded to elliptical in outline when viewed with the SEM (Figs. 5, 7). Cuticle was absent in in vitro leaves.
DISCUSSION
In vitro flowering: Not all plant species can easily flower in vitro. The utilisation of plant hormones is important to trigger in vitro flowering, besides carbon source, day length and lighting. So far in vitro flowering has been reported in species like tobacco, bamboo, orchids, Murraya paniculata etc. In species such as Lolium temulentum, a long day plant, the shoots could only flower in vitro when they were exposed to long hours of light. When the shoot explants were exposed to short day treatment, the shoots turned to vegetative phase of growth (McDaniel et al., 1991). The interaction
| Fig. 5: | Scanning electron micrograph of abaxial surface of in vivo leaf showing anomocytic stomata |
| Fig. 6: | Scanning electron micrograph of abaxial surface of in vitro leaf showing the distribution of the stomata and structure of trichome |
| Fig. 7: | Scanning electron micrograph of abaxial surface of in vitro leaf showing anomocytic stomata |
of sucrose and plant hormones, minerals and phenolic in in vitro flowering were also discussed in Torenia sp. (Tanimoto, 1981; Tanimoto and Harada, 1981).
| Fig. 8: | Scanning electron micrograph of abaxial surface of in vitro leaf showing the structure of the trichome |
| Fig. 9: | Light micrograph showing transverse section of in vivo lamina with cuticle (C), epidermis (E), palisade cells (P) and vascular bundle (V) |
| Fig. 10: | Light micrograph showing transverse section of in vitro lamina with epidermis (E), palisade cells (P) and vascular bundle (V) |
Hormones such as BA, NAA and GA3 (Zeevart, 1967) were often used to induce flowering. For instance, GA3 could induce in vitro flowering in Petunia hybrida grown in culture (Abdullah, 1998). However, GA3 could also inhibit in vitro flowering in Fuchsia hybrida (Sachs et al., 1967). In this study, M. paniculata was found to produce flowers under tissue culture system when 0.5-2.0 mg L-1 NAA was supplemented to the MS medium. However, flowering could also occur on MS basal. This could be due to endogenous hormones already present in the explants. BA was not effective in promoting in vitro flowering of this species, although BA was capable of giving rise to plant regeneration. Exogenous hormones including BA and NAA supplied to the medium and endogenous hormones already present in the explants can stimulate in vitro flowering in species such as tobacco (Pierik, 1967), but in this study, BA was totally inhibitory for in vitro flowering of Murraya paniculata.
Longer period of light (16 h) was favourable compared to shorter day length (8 h light). By subjecting the cultures to only 8 h light, in vitro flowering was reduced to almost half. This is in agreement with observations made by Pierik (1967) working on Lunaria annua. This could be due to the photosynthetic light affecting the overall growth of the plant; when more light available, the rate of photosynthesis will be higher, thus because flower initiation should be considered as part of growth process, all factors normally affecting growth also affect flower bud initiation.
Morphological and anatomical studiesCuticle: The presence and thickness of the cuticle is dictated by environmental factor. It is therefore more of ecological rather than of taxonomic importance. Wilkinson (1979) provides a list of environmental factors that affect cuticle thickness and notes that it is more frequently of value as an indicator of climate or habitat. The presence of cuticle in in vivo leaves and its absence in in vitro leaves has been expected since in vivo leaves is exposed to the sun.
Epidermis: The adaxial and abaxial epidermal cells of both in vitro and in vivo leaves vary from being isodiametric to irregular in shaped. The adaxial epidermal cells are larger than the abaxial epidermal cells. However, this is of no taxonomic significance since this characteristic is universal in the dicotyledons.
Mesophyll: The mesophyll in leaf transection showed a clear differentiation between a well-formed palisade and the adjacent spongy layer. Many studies on the ecological anatomy of tree species (Hanson, 1917; Ryder, 1954) indicate that the degree of mesophyll differentiation is highly dependent on the degree of exposure to the sun. According to them, leaves growing in sunshine have more palisade layers compared to leaves in shade and the palisade cells are long, large and close together. In leaves in heavy shade the palisade cells tend to become short, thin and loosely arranged. This may explain why in vivo leaves have biseriate palisade layers.
Vascular bundles: The vascular bundles are consistent in both in vivo and in vitro leaves as only arc-shaped vascular bundles were observed.
CONCLUSIONS
Longer period of light (16 h) was found to be more effective to trigger in vitro flowering of this species compared to short day. BA was inhibitory to in vitro flowering of this species.
The present study also suggest that the anatomical differences observed in both in vivo and in vitro leaves were due to the differences in the environment of the plants. Hence, when the in vitro plant is transferred to the soil, having the same environment as in vivo plant, no anatomical as well as morphological differences are to be expected.
ACKNOWLEDGMENT
The authors would like to thank the University of Malaya for the financial support of this work.