Jurnal Bioteknologi Pertanian Vol. 8, No.and 1, 2003, pp.

1-7 in Camellia sinensis Enhancement of somatic embryo , development plantlet recovery

Enhancement of somatic embryo development and plantlet recovery in Camellia sinensis by temporary liquid immersion
Meningkatkan perkembangan embrio somatik dan pembentukan planlet Camellia sinensis dengan sistem perendaman sesaat J.S. Tahardi1, Imron Riyadi1, and W.A. Dodd2
1

Biotechnology Research Institute for Estate Crops, Jalan Taman Kencana 1, Bogor 16151, Indonesia 2 University of Queensland, Gatton, Qld 4343, Australia

ABSTRAK
Perkembangan embrio yang tidak sinkron, perkecambahan yang rendah, dan pembentukan planlet yang tidak efisien merupakan kendala utama bagi penerapan embriogenesis somatik dalam perbanyakan massal tanaman unggul. Penelitian ini bertujuan untuk meningkatkan efisiensi pengembangan dan pendewasaan embrio somatik bagi pembentukan planlet teh ( Camellia sinensis L.) dengan sistem perendaman sesaat. Embrio somatik pada tahap globuler berkembang dengan cepat menjadi bentuk hati dan torpedo pada media cair woody plant (WP) yang diberi zat pengatur tumbuh (ZPT) BA 1 mg/l, IAA 5 mg/l, dan glutamin 100 mg/l setelah dikulturkan selama 4 minggu. Dalam dua siklus kultur berikutnya pada media yang mengandung BAP 1 mg/l + ABA 0,1 mg/l, dan BAP 0,1 mg/ l + ABA 0,5 mg/l, perkembangan embrio somatik meningkat frekuensinya ke fase torpedo dan kotiledon. Pendewasaan embrio dapat dicapai selama periode ini melalui perlakuan dehidrasi bertahap dan pengurangan nutrien dengan mengurangi frekuensi perendaman. Pada akhir fase (16 minggu), hampir semua embrio somatik mencapai fase torpedo dan kotiledon. Frekuensi perkecambahan embrio fase kotiledon menjadi tanaman yang lebih tinggi diperoleh setelah dipindahkan ke media WP padat tanpa ZPT. Perendaman sesaat menghasilkan perkembangan embrio yang sinkron, terutama dengan menekan terjadinya embriogenesis sekunder dan meregenerasikan embio somatik lebih cepat dengan frekuensi tinggi ke fase selanjutnya. Dibandingkan dengan teknik in vitro konvensional pada media padat, sistem perendaman sesaat lebih efisien dalam hal mengurangi subkultur dan upah kerja, sekaligus berpotensi untuk diterapkan dalam produksi bibit teh secara komersial. [ Kata kunci : Camellia sinensis , embriogenesis somatik, regenerasi in vitro ]

genesis for mass production of propagules from elite clones. A research was conducted to enhance the development and maturation of somatic embryos for efficient recovery of tea (Camellia sinensis L.) plantlets by using temporary immersion system (TIS). Globular somatic embryos developed rapidly into heart and torpedo stages in woody plant (WP) liquid medium supplemented with 1 mg/l BAP, 5 mg/l IAA, and 100 mg/l glutamine after 4 weeks of culture. During the next two culture cycles with medium replenishment and replacement of plant growth regulators (PGRs) by 1 mg/l BAP + 0.1 mg/ l ABA, and 0.1 mg/l BAP + 0.5 mg/l ABA, the development of somatic embryos progressed in increasing frequencies to torpedo and cotyledon stage. Maturation was achieved during this period by consecutive treatments of gradual dehydration and nutrient deprivation by reducing the frequency of immersion. By the end of 16 weeks, most of the somatic embryos had reached the torpedo and cotyledonary stages. Improved germination of the cotyledon-stage embryos and subsequent plant conversion were achieved after transfer to WP gelled medium without PGRs. Temporary immersion resulted in a synchronized embryo development, apparently through suppression of secondary embryogenesis, thus allowing rapid progression of somatic embryos in high frequencies into successive developmental stages. Compared to the conventional method of in vitro culture on gelled medium, the TIS protocol was more efficient, since it avoids culture transfer and reduces labour cost, and is potentially suitable for commercial production of tea propagules. [ Keywords : Camellia sinensis , somatic embryogenesis, in vitro regeneration]

INTRODUCTION Cultivated tea [ Camellia sinensis (L.) O. Kuntze] is conventionally propagated by single leaf-node cuttings. This method of production becomes limiting when large numbers are required from new clones of which only very few stock plants are available. Moreover, the process in establishing tea plants from cuttings is lengthy and labour-intensive, rendering it

ABSTRACT
Asynchrony in embryo development, low germination, and inefficient recovery of plantlets have been generally the major constraints precluding the use of somatic embryo-

2 ineffective for rapid dissemination of new clones for large-scale commercial plantings (Dodd 1994). Research on tea micropropagation has recently focused on exploring the potential of somatic embryogenesis as a more efficient means of plant multiplication and regeneration. Wachira and Ogada (1995), Ponsamuel et al. (1996), and Akula and Dodd (1998) reported induction of somatic embryogenesis and plant regeneration from cotyledonary and nodal explant cultures. However, in all these studies, little attention was given to the aspects of embryo development, maturation, germination, and conversion into plantlets. Synchrony of embryo development and maturation are necessary for efficient plant regeneration and production of propagules (Merkle 1995). A novel in vitro culture system based on temporary and periodic immersion of explants in a liquid medium has been tested successfully for synchronous development of embryogenic cultures of Hevea (Etienne et al . 1997), Citrus (Cabasson et al . 1997), oil palm (Tahardi, 1998), and coffee (Etienne-Barry et al. 1999). However, the somatic embryos developed by temporary immersion may sometimes show low capacity to germinate and poor plantlet conversion, as a result of insufficient embryo maturity (Cabasson et al. 1997). In horse chestnut, short periods of stress such as dehydration or nutrient starvation enhanced embryo germination and conversion into plantlets (Capuana and Debergh 1997). The objective of this research was to enhance the development of a culture method for improving embryo development and their conversion into tea plants using the temporary immersion system (TIS). Synchronization of embryo development and maturation was achievable after exposure of the TIS cultures to combinations of plant growth regulators (PGRs) and transient stress conditions.

J.S. Tahardi et al.

MATERIALS AND METHODS Studies on tea embryo development and maturation were carried out using two culture systems, namely TIS and gelled (control) cultures. Globular embryos of relatively uniform size (2-3 mm) were selected from proliferating embryogenic cultures grown on woody plant (WP) basal gelled medium supplemented with 2 mg/l BAP (Fig. 1a). The cultures were established from nodal explants taken from 8-year-old field-grown tea shrubs of clonal origin, following the sterilization protocol developed by Tahardi (1994) and the method for somatic embryo induction described by Akula and

Dodd (1998). The embryos were cultured in both systems for a total duration of 16 weeks, with changes of culture media and culture conditions every 4 weeks. The media used consisted of WP basal salt formulations (Lloyd and McCown 1980) supplemented with 100 mg/l glutamine and various PGRs (0.1-1.0 mg/l BAP, 5 mg/l IAA and 0.1-0.5 mg/l ABA) at specified culture stages and 30 g/l sucrose. The medium was adjusted to pH 5.8 and autoclaved at 121oC and 105 kPa for 20 minutes. Gelrite at 2 g/l was added for the gelled culture prior to autoclaving. The cultures were incubated at 25-27 o C under a 14-hour photoperiod with Philips cool white fluorescent tube (32 W) lighting providing an intensity of 30 mol/m2/s at the culture level. The TIS apparatus used in this study is basically a commercial liquid filter unit (Nalgene, Nalge Co., USA), modified by connecting the upper and the lower compartment (500-ml volume) by means of a small glass tube fitted to a screen disc at the bottom of the upper compartment (Fig. 1b). Samples of globular embryos averaging 200 in number and weighing about 4 g per sample were placed on a screen disc in the upper compartment, and about 125 ml of WP liquid medium in the lower compartment. When the lower compartment was pressurized, the nutrient solution was pumped from the bottom into the upper compartment. During the initial culture proliferation, which lasted for 4 weeks, immersion of the globular embryos was programmed at intervals of 6 hours for a duration of 3 minutes each. Gradual dehydration of the embryos was achieved by reducing the immersion frequency to 24 hours instead of every 6 hours. This cycle was continued for two consecutive periods of 4 weeks each, followed by a period (4 weeks) of starvation during which the nutrient solution was withheld from the embryos. At the end of each culture cycle, the TIS apparatus was disassembled for determination of biomass, developmental stage, and total number of embryos formed. Somatic embryos which had developed to the cotyledonary stage from both culture systems were allowed to germinate within 4 weeks on WP gelled medium without PGRs. Somatic embryos were considered germinated as soon as radicle emergence was observed. Germinated somatic embryos developed into plantlets on the same germination medium. As soon as a germinated somatic embryo developed an epicotyl followed by some leaf development, it was considered a plantlet. Determination of plantlet conversion or recovery frequency was carried out after another 4 weeks of culture on the gelled medium of the

Enhancement of somatic embryo development and plantlet recovery in Camellia sinensis

Fig. 1. Somatic embryo development and plant regeneration in Camellia sinensis (L.): (a) primary globular embryos; (b) somatic embryos proliferating and developing under temporary immersion (TIS) conditions; (c) somatic embryos at later stages showing synchronous maturation from TIS culture; (d) clumps of somatic embryos developing asynchronously on agar-gelled medium; (e) sequence showing progress of somatic embryo development and plant regeneration by TIS.

same composition. Experimental data were evaluated statistically using the Students t-test for each single treatment.

RESULTS AND DISCUSSION Embryo proliferation Previous studies in tea somatic embryogenesis have established the positive effect of TIS culture on embryo proliferation and development (Tahardi et al. 2000). In the present study, the effect of TIS was

further evaluated on tea embryo development, maturation, germination, and conversion into plantlets as influenced by different combinations of PGRs. Stress conditions which have been shown to enhance embryo maturation (Attree et al. 1991) were imposed in this study by reducing the frequency of liquid immersion from once every 6 hour to 24 hours or 4 weeks. As a control, globular embryos were cultured on the gelled medium of the same nutrient and phytohormonal composition as that of the TIS culture. Since the TIS medium underwent progressive deprivation of nutrients during the course of its culture, it was expected that any increase in biomass

4 and embryo production would be significantly less than that of the gelled culture where nutrient availability was not limiting. Such increases would be mostly associated with embryo growth and secondary embryogenesis respectively. Table 1 shows the effects of PGRs and immersion frequencies (stress conditions) on the development and maturation of tea somatic embryos in the TIS medium during a total culture period of 16 weeks. The culture sequence comprised four developmental phases: proliferation, development, maturation and further maturation, each lasting 4 weeks. At the start of the culture, 200 somatic embryos at the globular stage weighing 3.960 g per sample were used. At the end of the first culture or proliferation phase in the TIS system, globular embryos had increased by 88 in number (1.4-fold), and in biomass by 5.761 g (2.4-fold) in WP liquid medium supplemented with 1 mg/l BAP and 5 mg/l IAA. However, these increases were not significantly different from those attained in the gelled medium (control) where embryo production and biomass increased by 57 in number (1.3-fold) and 6.559 g (2.6fold), respectively. The comparable increase in embryo production and biomass after 4 weeks of culture in the two systems suggests that both proliferation and growth of somatic embryos were progressing at about the same rate. In the TIS culture, these proliferating embryos had also started to develop gradually into globular (72.2%), heart-shaped (16.4%), torpedo-shaped (8.9%), and cotyledonary embryos (2.5%) (Fig. 2a). In the gelled

J.S. Tahardi et al.

medium, however, the frequency of embryos remaining at the globular stage by the end of the proliferation phase was higher (80.2%), with much fewer embryos progressing to the torpedo (2.9%) and cotyledon (0%) stage (Fig. 2b). These results suggest that subsequent development from globular to cotyledonary embryos in tea was better facilitated by the TIS than by the gelled culture system, possibly through better aeration of the system. Embryo development Further development of somatic embryos in the TIS culture continued into the second culture (development) phase in the presence of 1 mg/l BAP and 0.1 mg/ l ABA, and under a reduced frequency of immersion (once every 24 hours). Biomass gain and embryo production during this development phase were moderately inhibited, apparently by the partial withdrawal of nutrients. By the end of 4 weeks or 8 weeks after culture initiation, embryo production had increased by 201 in number (1.7-fold), whereas biomass gain was only 5.121 g (1.5-fold) (Table 1). By this time, more globular embryos had progressed to the torpedo (19.7%) and cotyledon (12.7%) stages of development, with only 32% remaining as globular embryos (Fig. 1a). The increasing frequencies of torpedo and cotyledonary embryos at this stage suggest that the cultures had embarked upon the maturation phase. Both BAP and ABA at noninhibitory levels as applied here have been shown to

Table 1. Effects of plant growth regulators (PGRs) and immersion frequencies (stress conditions) on the production and growth of tea somatic embryos during different phases of culture in WP liquid (TIS) medium; the gelled medium served as a control. Culture phase and PGRs (mg/l) Immersion frequency (times) Proliferation BAP 1 + IAA 5 Development BAP 1 + ABA 0.1 Development and maturation BAP 0.1 + ABA 0.5 Further maturation PGRs-free Increase in Biomass (g) TIS 5.761 5.121* 7.755 0 Gelled 6.559 12.888 9.193 1.022 Number of somatic embryos TIS 88 201* 186* 57* Gelled 57 386 751 368

4 1 1 0

Initial number and fresh weight of globular embryos are 200 embryos per sample weighing 3.960 g. Each culture phase: 4 w; immersion frequency: once every 6 h (4x), 24 h (1x) or 4 w (0x) each for 3 min. Data represent means of three replicate cultures. Means within the rows followed by asterisks are significantly different (P < 0.05).

Enhancement of somatic embryo development and plantlet recovery in Camellia sinensis Frequency of SEs (%) 80 Proliferation Development Maturation Further maturation Proliferation Development Maturation Further maturation

Frequency of SEs (%) 80

60

60

40

40

20

20

Globular

Heart

Torpedo

Cotyledon

Globular

Heart

Torpedo

Cotyledon

Fig.2a. Development and maturation of tea somatic embryos (SEs) under temporary immersion system (TIS) and stress condition.

Fig.2b. Development and maturation of tea somatic embryos (SEs) on gelled medium (control).

be effective in fostering normal embryo maturation (Ammirato 1983). Our results are in line with the reported stimulatory and synergistic effects of BAP and ABA on embryo development and maturation in Norway spruce (Bozhkov et al. 1992). In the present study, embryo maturation appeared to be triggered or accelerated by a nutritional stress imposed as a result of partial withdrawal of nutrients from the TIS cultures, following a reduction in immersion frequency. A moderate increase in embryo production as observed here can probably be explained by a hypothesis that partial deprivation of nutrients suppressed normal proliferation of undifferentiated cells, thereby allowing embryogenic cell differentiation, leading to embryo production. In date palm cultures, enhancement of somatic embryogenesis was achieved after withholding sucrose for 2 weeks (Veramendi and Navarro 1996). On the other hand, in the gelled cultures, where no nutrient deprivation was imposed, the increase in biomass was significantly higher (12.888 g or 2.4-fold) than in the TIS culture, with a correspondingly higher production of secondary somatic embryos (386 in number) (Table 1). The continued supply of nutrients in the gelled cultures was apparently accountable for the significant gain in both biomass and secondary embryo production. However, this can not be construed as an advantage but rather an impediment to the synchronization of embryo development. Thus, fewer globular embryos were capable of maturing into the cotyledon stage (3.2%), even though the frequency of torpedo embryos was comparable to that of the TIS culture (Fig. 1b). The low frequency of

cotyledonary embryos in the gelled cultures suggests that embryo development was somehow arrested or inhibited at the preceeding stage. Embryo maturation Further development and maturation of somatic embryos in the TIS culture were evident during the third culture phase, when BAP was reduced to 0.1 mg/l and ABA increased to 0.5 mg/l. Somatic embryo production (186 in number or 1.4-fold) and biomass gain (7.755 g or 1.5-fold) were relatively stable as in the previous developmental phase (Table 1). However, more globular embryos developed into torpedo (23.0%) and cotyledonary embryos (14.1%), presumably in response to changes in phytohormonal concentrations and to the continued maintenance of cultures under reduced immersion frequencies (Fig. 1c and 2a). Exposure to a higher level of ABA during this phase appeared to be beneficial in accelerating the development of torpedo and cotyledonary embryos in liquid culture, in promoting embryo tolerance to water stress, and subsequently in enhancing their plant conversion efficiency (Attree et al. 1991). Our results confirmed the observation by Capuana and Debergh (1997) who reported improved viability, shoot elongation and plant conversion in horse chestnut somatic embryos exposed to slow desiccation and ABA. In the gelled cultures however, biomass gain (9.193 g 2.2-fold) and somatic embryo production (751 in number or 2.2-fold) via secondary embryogenesis still persisted, suggesting that embryo proliferation and growth had not ceased completely, even though the

6 cultures had embarked upon the development and maturation phase. The effect of ABA on embryo maturation in the gelled culture was consequently less pronounced, as indicated by a majority of somatic embryos (81.8%) still remaining in the globular (38.0%) and heart (43.8%) stage, and very few in the torpedo (15.5%) and the cotyledonary (2.7%) stage, even after the third culture phase (Fig. 2b). This shows that under the gelled culture conditions, synchronization of embryo development and maturation had not been fully achieved as in the TIS culture. Further embryo maturation During the fourth culture phase, complete withdrawal of nutrients from the TIS culture exerted a dramatic effect on the subsequent development of tea somatic embryos. Embryo differentiation and maturation appeared to have been accelerated, bringing more somatic embryos to the torpedo (25.9%) and cotyledonary stage (20.6%), with fewer remaining as globular embryos (10.8%). Again, the promotional effect of nutrient deprivation on embryo maturation as observed here can be explained by the earlier hypothesis of stress induction. As expected, there was absolutely no gain in biomass after the starvation treatment, even though there was still a small increase (368 in number or 1.1-fold) in embryo production via secondary embryogenesis (Table 1). A similar trend of embryo development leading to torpedo and cotyledonary somatic embryos was also evident in the gelled cultures without PGRs. However, their development was much less synchronized, as numerous embryos were aggregated (Fig. 1d) and still arrested at the globular stage (24.1% vs 10.8% in TIS) (Fig. 2b). In addition, there was still a slight increase in biomass (1.022 g or 1.03-fold) and embryo production (368 in number or 1.3-fold) in the gelled cultures, where nutrient deprivation was not imposed. Embryo germination and plantlet recovery Culture of cotyledonary embryos of tea derived from TIS conditions on gelled medium for 4 weeks showed a higher frequency of germination response (46.4%) than those derived from gelled medium without stress conditions (25.4%) (Table 2). Similarly, the rate of plant conversion from germinated tea embryos (8 weeks after transfer) was consistently higher for those derived from TIS (87.7%) than from gelled culture (38.3%). Shoot development in the latter was often followed by some basal stem callusing which inhibited root elongation (Fig. 1e).

J.S. Tahardi et al. Table 2. Effect of culture system and culture conditions during tea embryo development on subsequent embryo germination and plantlet recovery 8 weeks after transfer to a gelled medium. Culture system Temporary immersion Gelled medium Number of cotyledonary embryos 345 181 Plantlet Germination conversion (%) (%) 46.4* 25.4 87.7* 38.3

Data represent means of three replicates for each culture system. Means within the columns followed by asterisks are significantly different (P < 0.05).

Dehydration and nutrient deprivation imposed by the TIS conditions employed in the present study proved to be highly effective not only in promoting embryo development and maturation but also in enhancing germination and subsequent plant conversion. Exactly how this was achieved in the case of tea discussed here is beyond the scope of this paper but reference may be made to Krikorian (2000) for an in-depth discussion of various factors, including hyperhydricity, which affect somatic embryo development. Indeed, stress conditions, such as high sucrose and high agar content, application of ABA, and partial or complete withdrawal of nutrients from culture media have been shown to induce the accumulation of storage products in both zygotic and somatic embryos during embryo maturation (McKersie et al. 1995). It has been suggested by Etienne et al . (1993) that the availability of starch and protein reserves in the cotyledonary tissues increases the vigour of somatic embryos, and stimulates the formation of root and shoot meristems during the germination of Hevea somatic embryos. CONCLUSIONS An effective protocol for relatively synchronous development of tea somatic embryos and subsequent plant recovery using TIS has been established. Optimization of embryo maturation which is essential for attaining a high frequency of germination and plantlet conversion was achieved through sequential manipulations of culture conditions and media. Application of BAP in conjunction with ABA along with gradual withdrawal of nutrients from the TIS cultures promoted a relatively synchronized development and maturation of tea somatic embryos, leading to improved germination and plant recovery. Com-

Enhancement of somatic embryo development and plantlet recovery in Camellia sinensis

pared to the conventional method of in vitro culture on gelled medium, this protocol is more efficient, since it avoids culture transfer and reduces labour cost, and is potentially suitable for commercial production of tea propagules. ACKNOWLEDGEMENTS The authors acknowledged with gratitude the financial support provided by the Australian Centre for International Agricultural Research (ACIAR), Canberra, for this work. Thanks are also due to Prof. Dr. Abraham D. Krikorian, Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York, USA for critically reading the manuscript. REFERENCES
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