Reliable normalization and accurate quantification are essential for gene expression studies in oil palm mesocarp. This study assessed three candidate reference genes (Actin, GAPDH, and EF1α) for stability using four algorithms (ΔCt, BestKeeper, NormFinder, and GeNorm). Actin was consistently identified as the most stable single housekeeping gene (HKG) (stability value 0.021), while the combination of GAPDH and EF1α provided the most stable normalization pair (stability value 0.017), supporting their use as internal controls for normalization. Normalized expression analysis of two key biosynthetic genes, acetyl-CoA carboxylase (ACCase) and phytoene synthase (Psy), was conducted in contrasting phenotypes differing in oil yield and carotene content. The high-yielding, high-carotene genotype displayed lower ΔCt values for both ACCase (4.67 vs. 6.19) and Psy (4.75 vs. 5.26), corresponding to 2.86-fold and 1.42-fold higher expression, respectively. These expression differences aligned closely with phenotypic outcomes, as the superior traits achieved a 40.8% increase in oil yield and a 155.3% increase in carotene content. Expression patterns showed stage-specific specialization: ACCase was upregulated during the unripe stage, enhancing fatty acid biosynthesis, while Psy was most differentially expressed during ripening, coinciding with carotenoid accumulation. Together, these findings suggest that transcript abundance of oil and carotenoid pathways may be coordinated in elite oil palm and provide a framework for linking gene expression with metabolic output, which may inform future efforts to improve oil yield and quality

The rising global demand for palm oil necessitates innovative strategies to accelerate yield improvement and ensure sustainable production. Conventional propagation methods are limited by long regeneration cycles, making tissue culture a more practical alternative that relies on elite ortet identification. This preliminary study aimed to evaluate crude palm oil quality, leaf metabolite profiles, and gene expression as early indicators for selecting elite ortets. Mature fruits were used to assess oil yield, free fatty acid, and β-carotene contents, while leaves were analyzed for chlorophyll and total phenolic content. Based on biochemical screening, three candidate ortets were further evaluated for the expression of acetyl-CoA carboxylase (ACCase) and phytoene synthase (PSY) genes. Oil yield ranged from 77-83%, FFA 5-9%, and β-carotene 200-450 mg kg-1 CPO across candidates. Candidate 3.7 exhibited the highest oil yield and elevated ACCase expression, while candidate 6.12 showed the highest β-carotene content and PSY expression. Candidate 5.7 demonstrated relatively low FFA and favorable chlorophyll levels. These results suggest an association between ACCase expression and oil yield, as well as between PSY expression and β- carotene accumulation. Collectively, these findings support a proof-of-concept for an integrated profiling strategy to select elite ortets for advanced tissue culture propagation.

In vitro embryo cultivation is the principal method for reproducing the kopyor coconut, an indigenous Indonesian species. Acclimatization is a critical step in this approach. The purpose of this study was to improve the survival and development rate of kopyor coconut seedlings grown in vitro during the acclimatization phase by using biological, organic, and mixed organic-biological biostimulants. This study employed a completely randomized block design that comprised the inclusion of biological, organic, and mixed biological-organic biostimulants, with no biostimulant as a control. The biological stimulant employed in this study was arbuscular mycorrhizal fungus (AMF), while the organic biostimulant was seaweed extract. Each treatment had 50 plantlets in triplicate. The results demonstrated that the treatment of biological and organic biostimulants increased plantlet survival rates during acclimatization, beginning 2 months after application and remaining consistent for 4 months. The biological stimulant application produced the highest plantlet survival rate (>94%). Based on how quickly the plantlets transitioned to the next stage, it was discovered that after 2-months incubation, most of the plantlets had already transferred to the second phase of acclimatization (opened tunnel). After 4-months incubation, the biological stimulant treatment produced the most plantlets at the later stage. However, throughout the 4-month acclimation period, more plantlets were transported from the pre-nursery to the main nursery, particularly with the use of organic biostimulants (56-64%). Keywords: arbuscular mycorrhizal fungi, plantlet, survival rate, seaweed

Kopyor coconut (Cocos nucifera var. Kopyor), a native Indonesian coconut, differs from common coconuts because it has brittle flesh (endosperm) due to a natural genetic mutation. Kopyor coconut seedlings have been successfully cultivated from the tissue culture technique, which can produce kopyor above 99%. Nowadays, the demand for kopyor continues to increase. The cultivation of kopyor is centralized in several locations in Indonesia. Hence, the time of kopyor distribution to consumers is crucial in the kopyor business. This study evaluated changes in the characteristics of whole fruit and peeled kopyor coconuts during distribution. The observations included quality attributes (appearance, aroma, texture, and taste), physical observations (pH and water content), and microbial observations using the total plate count method. Whole kopyor showed better quality attributes than peeled kopyor. Peeled kopyor coconut flesh began to change color to yellowish-white, mushy, and had a rancid smell and a sour taste in the second week, while whole fruit kopyor in the fourth week. In conclusion, the distribution of peeled kopyor should be carried out in less than two weeks, or even sooner, while whole fruit kopyor can be distributed for up to two weeks without altering the characteristic taste of kopyor.

The utilization of temporary immersion system-bioreactors (TIB) has shown promise for sugarcane micropropagation due to its capacity for high-yield seedlings production. However, the temporary immersion system also carries the risk of somaclonal variations (SV) in regenerated plants. SV, encompassing phenotypic changes resulting from genetic or epigenetic alterations, and it presents challenges in preserving the genetic uniformity of the seedlings, a crucial factor for sugarcane productivity. A molecular marker like Simple Sequence Repeats (SSR) offers a reliable means to assess the genetic stability of TIB-derived sugarcane seedlings, ensurinng that the temporary immersion system-based micropropagation produces true-to-type planting materials. In this study, SSR analysis was conducted to determine the relative genetic stability of TIB-derived sugarcanes, using 15 SSR markers, generating over 200 DNA fragments ranging from 150 to 4,000 bp in size. The genetic stability was calculated as the Jaccard’s similarity index of the SSR patterns of TIB-derived sugarcanes compared to their mother plants. The genetic stability varied between 92 and 96% in three Indonesian sugarcane varieties (PSKA 942, PS 094, and PS 091) which is relatively high considering the high cell mass multiplication in TIB (up to 30 multiplies). Morphological observations in the field also revealed no differences between TIB-derived sugarcane and their mother plants, convincing the true-to-type of micropropagation products. This study demonstrated the high relative genetic stability of TIB-derived sugarcanes, paving the way for large-scale commercial application in plantations.

Indonesia has released some elite varieties of sugarcane that are suitable for Indonesian climatic and land conditions. Therefore, large quantities of these elite sugarcane seedlings are needed to support the Indonesia's sugar self-sufficiency programs. The large quantities seedlings can be provided through scaling-up in vitro propagation method using bioreactors, such as Temporary Immersion Bioreactor (TIB). Here, we conducted a study to analyze the response of different plant genetic materials on the in vitro propagation of some Indonesian elite sugarcane varieties using TIB. The sugarcane varieties used for the study were the rainfed sugarcane varieties (PSKA 942, PS 094) and the irrigated sugarcane varieties (PS 091, PS 881), released by the Indonesian Sugar Research Institute. The plant materials used as explants for in vitro propagation were the young spindle leaves isolated from the plant canes. The in vitro propagation stages were explants preparation and sterilization, callus initiation, callus proliferation and regeneration in TIB, shoot maturation, and acclimatization. Results showed that all sugarcane varieties have been successfully induced to form callus ranged 11.6 – 33.5%, proliferate and regenerate in TIB. Proliferation rates in TIB ranged 4.45 – 33.76 multiplies of biomass after two proliferation cycles. However, in general, the rainfed sugarcane varieties showed better response, growth, and regeneration rate than the irrigated sugarcane varieties. The in vitro-derived sugarcane seedlings have been successfully obtained from all varieties except for PS 881, with the highest shoots production was PSKA 942 (147.7 shoots/flask).

Genome editing through cisgenesis develops into scientific breakthroughs in accelerating oil palm breeding programs. However, one remaining problem is the low success of transformed-calli regeneration, while its scientific explanation is still underexplored. This study aimed to characterize and regenerate transformed-calli using various amino acids and antioxidants. Transformed callus that did not regenerate (un-regenerated transformed callus or UTC) after the transformation process was taken, then T-DNA integration was detected using the NPTII gene. Furthermore, the UTC was divided into four types based on morphological characteristics. The four types of UTCs were regenerated on media enriched with glutamine (for Type-1 callus), cysteine and putrescine (for Type-2 callus), and a combination of cysteine and ascorbic acid (for Type-3 and Type-4 callus). The research results obtained NPTII successfully amplified with a band size of 700bp. The results showed that on Type-1 callus, enrichment media with 10 mg L-1 L-glutamine could induce the formation of new nodular structures on UTC Type-1. On Type-2, media enriched with 5 mg L-1 L-cysteine + 20 mg L-1 putrescine increased the density of callus structures. Media enriched with 25 mg L-1 ascorbic acid + 25 mg L-1 L-cysteine could prevent the spread of brown callus on Type-3 callus, while Type-4 callus did not show any response and became dry. Our new findings will facilitate the basic research and unregenerated transformed callus and morphological callus development behavior in oil palm.

Abstract Browning on in vitro culture is one of the problems in somatic embryo regeneration that leads to growth inhibition and death of explants. This study aimed to prevent and control browning of oil palm somatic embryos (SE) in the Temporary Immersion System (TIS). The research was conducted in two stages; the first stage was applying an antioxidant mixture at concentrations of 20, 50, and 100 mg L-1 on the browning palm oil SE in TIS. The second stage was carried out by modification of the basal media, namely DF (control), MS, SM, and SMT. The explants used in experiment 2 were browning and fresh SE. The results showed that using 50 ppm antioxidants mixture increased the formation of new SE by up to 15 embryos per flask. Applying SMT basal media on browning SE could decrease browning rate sharply after two cycle cultures by up to 60% browning rate reduction. In this media, it was also seen that the number of new SE increased by up to 250 embryos and produced 15 germinants, while in control, only 60 SE and five germinants were formed. Whereas using SMT basal medium on fresh embryos could prevent browning and increase the formation of new embryos and germinants. In conclusion, using 50 ppm antioxidants can stimulate the formation of new embryos. Using SMT basal media could prevent browning and overcome the occurrence of browning in embryos by increasing the formation of new embryos and germinants.

Laboratorium kultur jaringan memiliki peran dalam industri perkebunan kelapa sawit, salah satunya yaitu untuk produksi bibit klonal skala massal. Produksi bibit klonal kelapa sawit umumnya dilakukan melalui teknik embriogenesis somatik yang melibatkan beberapa tahap. Seluruh rangkaian proses produksi tak terlepas dari alur pendataan yang memastikan proses berjalan dengan benar dan hasil produk akurat. Penerapan kode QR dalam pendataan di laboratorium kultur jaringan, seperti dalam produksi bibit klonal kelapa sawit, memberikan keuntungan dengan menggantikan pendataan manual yang rentan terhadap kesalahan manusia dan waktu yang tidak efisien. Aplikasi pendataan berbasis kode QR, seperti Tissue Culture Tracking membantu dalam pelabelan digital dan pemantauan proses, yang memungkinkan penelusuran data yang akurat serta meningkatkan efisiensi produksi bibit klonal. Beberapa tantangan dalam penerapan digitalisasi data dalam laboratorium kultur jaringan antara lain akses internet yang memadai, kompatibilitas perangkat, serta keterampilan dan budaya kerja personel. Revitalisasi fasilitas, pelatihan, dan pembiasaan adalah solusi agar digitalisasi pendataan berbasis kode QR dalam laboratorium kultur jaringan dapat diimplementasikan dengan baik.

The Zambli variety of date palm shows potential for cultivation in tropical regions, as its fruits are edible during the Rutab stage. However, large-scale production of Zambli seedlings presents a significant challenge. In vitro propagation offers a solution for producing large quantities of clonal planting material. This study focuses on inducing callus formation from the four-layered shoot tips of young leaves and regenerating these calli into plantlets through somatic embryogenesis. Explants were cultured on a modified MS medium with 10, 50, or 100 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), combined with 1 or 3 mg L-1 N6-(2-isopentenyl)adenine (2-iP). Embryo maturation was performed on the same medium without 2,4-D, while a hormone-free medium was used for plantlet regeneration. The results indicated that the highest callus induction occurred from the younger leaf layer (layer 1) in the medium containing 100 mg L-1 2,4-D and 1 mg L-1 2-iP, achieving a callus formation rate of 82.3%. Successful callus induction was achieved from the first, second, and third layers of young leaves. Somatic embryo maturation and plantlet regeneration were also completed, producing vigorous, well-rooted plantlets. Additionally, the development of date palm cv. Zambli in vitro culture through somatic embryogenesis was confirmed through histological analysis.

Optimization of in vitro shoot growth is necessary to shorten the culture time and produce vigorous oil palm plantlets. This research was conducted to determine the best media and culture techniques to accelerate in vitro shoot growth of oil palm. Shoots of oil palm derived from somatic embryogenesis were grown on DF media with two culture systems (solid medium and double-layer) combined with hormone treatments (0.5-1 mg L-1 giberellin/GA3 and 0.5 mg L-1 Benzyl amino purine/BAP or thidiazuron/ TDZ). Further optimization was conducted using different bottle closures (polypropylene screw caps and plastic wraps) and macronutrients (standard or double concentrations). This research was conducted using a completely randomized design (CRD), with each treatment consisting of 5 bottle replications and each bottle consisting of 5 shoots. The results showed that media with double-layer system combined with GA3 (0.5-1 mg L-1) and TDZ (0.5 mg L-1) gave the highest shoot height increment. The use of double-strength macronutrient media combined with screw bottle caps increased shoot height (136-167 %) and decreased shoot tip necrosis (0-24 %). Plastic wrap bottle caps increased shoot tip necrosis (STN), while doubling macronutrients reduced STN. The growth of oil palm shoots began to slow down after 5 weeks of culture. In conclusion, the optimal conditions for in vitro shoot growth of oil palm were at usage of double-layer media added with GA3 0.5-1 mg L-1, TDZ 0.5 mg L-1, and double macronutrients on bottle jars with polypropylene screw caps.

Contamination in the in vitro culture is a critical problem causing the failure of seed production. Contamination in the oil palm plantlet is detrimental, considering that oil palm propagation is difficult and takes a long time. This research aimed to study the effect of sterilization during acclimatization of the contaminated oil palm plantlets by fungi on viability and to determine the optimum viability achieved from the contaminated materials. The materials used were contaminated plantlets of oil palm with roots, four leaves, and a height of about 17 cm. The plantlets were removed from the tube and cleaned with running tap water, then were sterilized, with treatments P1: soaking in benomyl-mancozeb-sodium hypochlorite and mannitol and rinsing with aquadest, P2: soaking in benomyl-mancozeb, P3: soaking in mancozeb. Cleaning plantlets under running tap water was carried out as a control treatment. The results showed that at 10 weeks after acclimatization, the survival rate of plantlets in each treatment (P1, P2, and P3) was significantly higher than that of the control. Sterilization methods affect the time new leaves emerge, leaf condition after sterilization treatment, and shoot height. The lowest fungal contamination after treatments was found in P2, followed by P3. After 3 months, plantlet survival rate decreased, with the highest survival rate in treatment P3 (32.3%) followed by treatment P2 (22.5%). In conclusion, acclimatization of contaminated oil palm plantlets can be carried out using a suitable sterilization treatment. Sterilization affects the survival rate and growth of in vitro-contaminated oil palm plantlets during acclimatization.

AbstrakBioreaktor perendaman sesaat (BPS) telah digunakan secara luas untuk propagasi skala massal berbagai tanaman penting, termasuk tanaman tebu. BPS menyediakan sistem kultur semi-otomatis dan kondisi optimal bagi pertumbuhan tanaman. Beberapa faktor menentukan pertumbuhan tanaman pada BPS, salah satunya densitas dari eksplan. Oleh karena itu, penelitian dilakukan untuk menentukan bobot awal yang optimal untuk kalus tebu yang dikulturkan pada BPS, serta mengevaluasi pengaruh perbedaan bobot awal kalus tersebut terhadap proliferasi dan regenerasi kalus tebu. Kalus tebu diinduksi dari daun muda yang masih menggulung dari empat varietas tebu unggul Indonesia. Bobot awal kalus yang dikultur ke dalam bejana TIB yaitu 0,05 g; 0,1 g; 0,2 g; 0,5 g; dan 1,0 g untuk setiap bejana. Kalus kemudian melalui tahap proliferasi pada BPS sebanyak tiga siklus, kemudian kalus diregenerasi pada BPS dengan perlakuan auksin dan sitokinin. Hasil penelitian menunjukkan bahwa 0,2 g merupakan bobot awal kalus yang efisien untuk proliferasi kalus tebu pada TIB, dimana eksponensial multiplikasi kalus tercapai pada bobot awal tersebut, yaitu untuk masing-masing varietas 130,3 kali (PSKA 942), 136,8 kali (PS 094), 21,3 (PS 881), dan 12,9 kali (PS 091) setelah 12 minggu. Densitas kalus pada TIB berkorelasi negatif dengan karakteristik fisikokimia medium. Hal ini menggambarkan variasi intensitas pertumbuhan dan metabolisme kalus dengan adanya perbedaan densitas pada BPS. Penggunaan BAP 0,2 mg L-1 bersama kinetin 0,2 mg L-1 paling sesuai untuk memacu regenerasi kalus tebu dengan menghasilkan jumlah tunas terbanyak dalam waktu relatif lebih cepat (1 – 2 minggu lebih cepat) dibandingkan perlakuan lainnya dan dengan tingkat kejadian pencoklatan yang rendah.[Kata kunci: kultur in vitro, kultur cair, proliferasi]AbstractTemporary immersion bioreactor (TIB) has been utilized for the mass-scale propagation of many important plants, including sugarcane. TIB facilitates a semiautomated culture system and provides optimal conditions for plant growth. Several factors determine plant growth in the TIB, such as explant density. Therefore, an experiment was carried out to determine the optimal initial weight of sugarcane calli and to evaluate its effect on the proliferation and regeneration in TIB. Sugarcane calli were induced from spindle leaves isolated from four Indonesian prime sugarcane varieties. The initial weights of the calli cultured in the TIB flasks were 0.05 g, 0.1 g, 0.2 g, 0.5 g and 1.0 g per flask. The calli were proliferated through three cycles in TIB, and subsequently regenerated in TIB with auxin and cytokinin treatments. The results of the experiments showed that 0.2 g was the most efficient initial weight for sugarcane callus proliferation in the TIB, resulting in an exponential multiplication rate of 130.3-fold (PSKA 942), 136.8-fold (PS 094), 21.3-fold (PS 881), and 12.9-fold (PS 091) within 12 weeks. In the TIB, callus density showed a negative correlation with the physicochemical properties of the medium, demonstrating various growth intensities or metabolic activities of calli at different densities in the TIB. The use of 0.2 mg L-1 BAP along with 0.2 mg L-1 kinetin was suitable for promoting the regeneration of sugarcane calli and producing the highest number of shoots in a relatively short amount of time (1 – 2 weeks faster) with low incidences of browning.[Keywords: in vitro culture, liquid culture, proliferation]

Sweet potato (Ipomoea batatas L.), a tuber-producing plant, is a functional food that produces carbohydrates while meeting nutritional needs. Propagation of sweet potato through tissue culture is often hampered in the initial culture due to contamination. The study aimed to optimize sterilization of explants and growth of in vitro culture of purple sweet potato. Tubers of purple sweet potato cv. Antin 2 were ex vitro cultured through a semi-immersion system to produce shoots. The shoots as explants were sterilized with alcohol, fungicide, and sodium hypochlorite (P1); alcohol and sodium hypochlorite (P2); sodium hypochlorite (P3), and washed on sterile distilled water as control (C). The explants were then cultured on MS solid medium in tubes and jars. Growth and multiplication of shoots were carried out on MS solid medium added with cytokinins (BA and kinetin) at different concentrations. The results showed that the best sterilization method was obtained in the sodium hypochlorite (P3) and alcohol-sodium hypochlorite (P2) treatments, with sterile shoots reaching 100%. Planting the explants in jars gave higher normal shoot formation (85–100%) than that in tubes (12.5–48%). The use of kinetin at 0.5–1 mg L-1 gave good shoot vigor. The best axillary shoot multiplication was found on media with 0.5 mg L-1 BA. Growing explants on the semi-immersion system and sodium hypochlorite sterilization produced the highest sterile ones, whereas culturing three explants promotes normal growth straight after sterilization.

The leaves of sweetener plant Stevia rebaudiana contain secondary metabolites of steviol glycosides which are very sweet, with no calorie and zero glycemic index. Propagation of stevia by seeds is ineffective due to its low germination rate and diverse progenies. The tissue culture of stevia can be used to mass propagate rapidly and is commonly conducted by shoot multiplication. Up to now, the technology of somatic embryogenesis (SE) in stevia has not been successful yet. SE is developed to increase the production scale, rejuvenate clonal-propagated plants, and plant genetic transformation. The research objective was to develop protocols for the initiation, proliferation, and development of embryogenic calli of stevia as potential materials for SE. The explants used were young leaves, nodes, and internodes of axenic plantlets of stevia BX clone. The explants were cultured on MS solid media containing different concentrations of auxin and cytokinin for callus initiation. Callus emerged after 2-3 weeks of culture. The calli obtained were proliferated by subculturing several times as material stocks for indirect SE. MS solid media added with 1 µM 3,4-D and 16 mM CaCl2 gave the highest callus multiplication rate (4.7 times in 3 weeks). The selection of embryogenic calli was made continuously to obtain a pure line of embryogenic calli. Three types of calli attained were friable, fast-growing, yellowish calli, shiny nodular calli, and greenish nodular calli. Histological studies revealed that cells of the nodular calli had been differentiated to potentially formed somatic embryos.

The leaves of sweetener plant Stevia rebaudiana contain secondary metabolites of steviol glycosides which are very sweet, with no calorie and zero glycemic index. Propagation of stevia by seeds is ineffective due to its low germination rate and diverse progenies. The tissue culture of stevia can be used to mass propagate rapidly and is commonly conducted by shoot multiplication. Up to now, the technology of somatic embryogenesis (SE) in stevia has not been successful yet. SE is developed to increase the production scale, rejuvenate clonal-propagated plants, and plant genetic transformation. The research objective was to develop protocols for the initiation, proliferation, and development of embryogenic calli of stevia as potential materials for SE. The explants used were young leaves, nodes, and internodes of axenic plantlets of stevia BX clone. The explants were cultured on MS solid media containing different concentrations of auxin and cytokinin for callus initiation. Callus emerged after 2-3 weeks of culture. The calli obtained were proliferated by subculturing several times as material stocks for indirect SE. MS solid media added with 1 μM 3,4-D and 16 mM CaCl2 gave the highest callus multiplication rate (4.7 times in 3 weeks). The selection of embryogenic calli was made continuously to obtain a pure line of embryogenic calli. Three types of calli attained were friable, fast-growing, yellowish calli, shiny nodular calli, and greenish nodular calli. Histological studies revealed that cells of the nodular calli had been differentiated to potentially formed somatic embryos.

The cultivation of date palm in Indonesia has increased since the last decade. However, the superior date palm seedlings are still limited and most of them are imported from other countries. The mass supply of superior date palm seedlings can be provided by in vitro propagation in the bioreactor. Therefore, the research was conducted to develop a protocol of date palm in vitro propagation by using Temporary Immersion Bioreactor (TIB). The in vitro propagation was carried out through somatic embryogenesis technique using meristematic tissues isolated from offshoots of date palm female clone cv. Zambli as explants. The explants were sterilized and then cultured to produce embryogenic calli and somatic embryos. Afterwards, somatic embryos germination and plantlets formation were conducted in TIB with treatments of immersion period: 3, 10, and 30 minutes every 6 hours, with 8 replications, The results showed that the optimal somatic embryo germination in TIB was with the immersion period of 30 min every 6 h, resulting in the most formation of shoots and fresh biomass weight increment up to nearly threefold in 6 weeks. Thereafter, plantlets formation in TIB with immersion period of 10 min and 30 min every 6 h exhibited similar performances in producing more plantlets with higher total fresh weight and better vigor than those of 3 min every 6 h. However, there were more rooted plantlets in the TIB with immersion period of 10 min every 6 h. Based on the results, an in vitro propagation protocol via somatic embryogenesis in TIB has been successfully developed for mass propagation of date palm cv. Zambli, which produced plantlets with good vigor and rooting.

Stevia rebaudiana Bertoni, a sweetener plant, has been cultivated in Indonesia since the early 1980s, but the yield is lower than in other countries. Five selected introduced stevia clones were planted at Megamendung, Bogor, Indonesia (6°39’ S, 106°56’ E, 800 m above sea level) on December 2014 until November 2015 to evaluate their growth and biomass yield. The growth and yield were observed from 6 to 12 months after planting. In addition, stevioside, rebaudioside A (reb A), and total steviol glycoside (TSG) contents of leaves were measured using high performance liquid chromatography (HPLC). At six months after planting, stem diameter was 8-11 mm, plant canopy diameter was 23-30 cm, plant height was 24-35 cm, and plant mortality was less than 5% except for clone BU at 30.7% due to Sclerotium attack. The plants flowered at different times, therefore had different harvest durations, from 4 weeks (clone BM) to 8 weeks (clone BP). The highest leaf yield was clone BM (6.04 ton ha-1 per year), followed by BX (4.91 ton ha-1 per year), and BP (4.38 ton ha-1 per year). From the five clones tested, clone BM was the best for leaf, TSG, and stevioside yields; whereas clone BS and BP were the best for reb A yield. Keywords: rebaudioside A, Stevia rebaudiana, stevioside, total steviol glycoside

Aklimatisasi merupakan masa transisi sebelum kultur in vitro dapat ditanam di lingkungan ex vitro. Di daerah tropis, stevia seyogianya ditanam di dataran tinggi. Pengembangan klon stevia yang sesuai untuk dataran rendah di kawasan tropis sangat penting untuk memungkinkan penggunaan mekanisasi pada pertanaman stevia yang luas. Tujuan penelitian ini adalah menentukan pengaruh kondisi eksplan dan periode hardening terhadap daya hidup dan pertumbuhan pada tahap aklimatisasi dan pertumbuhan awal stevia klon BS 22 pada area terbuka di dataran rendah di wilayah tropis. Penelitian pertama dilangsungkan menggunakan umur tunas yang berbeda: 0, 1, 2, dan 3 minggu yang dikultur pada media padat sebagai sumber bahan eksplan. Penelitian kedua menggunakan satu buku stevia pada periode hardening dalam media cair selama 1, 4, 7 dan 10 hari. Aklimatisasi dilaksanakan dengan menanam eksplan dalam medium tumbuh campuran pada multi-tray dan diletakkan di dalam sungkup plastik tertutup selama 1 bulan. Tanaman yang berhasil hidup kemudian dipindah ke polibeg pada area terbuka dengan sinar matahari penuh. Pengamatan daya hidup dan pertumbuhan dilakukan pada akhir tahap aklimatisasi dan setelah 2 bulan di area terbuka. Hasil penelitian menunjukkan bahwa eksplan umur 1 minggu yang dikultur pada media padat mempunyai daya hidup tertinggi yakni 83%. Buku tunggal stevia yang dikultur pada medium cair pada tahap hardening selama 4 hari meningkatkan daya hidup menjadi 97% selama aklimatisasi 1 bulan. Setelah aklimatisasi, tinggi tanaman secara rata-rata adalah 2,6 cm dengan 10,6 helai daun. Tanaman yang dipindah ke area terbuka tumbuh pesat dengan tinggi tanaman mencapai 12 cm dengan 30 helai daun dan daya hidup 63% setelah 2 bulan. Hal ini menunjukkan bahwa stevia klon BS 22 mungkin sesuai untuk dataran rendah di daerah tropis. [Kata kunci: tanaman pemanis, eksplan tunas, tahap hardening, daya hidup, tropika] AbstractAcclimatization is a transition period before in vitro culture can be planted in ex vitro environment. In the tropical region, stevia is should be planted at high altitude areas. The development of stevia clones suitable for low land area in the tropics is important to make it possible to apply mechanization in a large scale stevia plantation. The purpose of this research was to determine the effect of explant conditions and hardening period on survival rate and growth during acclimatization stage and early growth of stevia clone B 22 in an open area at low altitude area in the tropics. The first experiment was conducted using different shoot ages: 0, 1, 2 and 3 weeks cultured on solid media as an explant material source. The second experiment was using single node of stevia in different hardening periods in liquid media for 1, 4, 7 and 10 days. Acclimatization was carried out by planting the explants on a mixture growing medium in multi-trays and placed inside a closed plastic tunnel for 1 month. The survival rate and growth parameters were observed at the end of acclimatization stage and after 2 months in the open area. The results show that 1-week explant age on solid media had the highest survival rate at 83%. Hardening single node of shoot in a liquid medium for 4 days increased the survival rate to 97% in 1 month acclimatization stage. After acclimatization, the plant height on average was 2.6 cm with 10.6 leaves. The survived plants planted in an open area grew rapidly to 12 cm in height with 30 leaves and survival rate 63% within 2 months. It indicates that stevia clone BS 22 may suitable for a low altitude area in the tropics. [Key words: sweetener plant, shoot explant, hardening period, survival rate, tropics]