Plantlet Formation Research Articles

Evaluation of host status of garlic varieties for a plant-parasitic nematode, Ditylenchus destructor, by using in vitro inoculation.

Ditylenchus destructor is a plant-parasitic nematode that severely damages garlic (Allium sativum L.) in Japan. D. destructor is detected in roots, bulbs, and outer bulb skins of garlic at harvest; however, the resistance of garlic to D. destructor infection is not well understood. Here, we investigated the propagation of D. destructor in storage organs and roots using in vitro plantlets of six Japanese garlic varieties to exclude the effects of microbes and to uniform growing conditions. In vitro inoculation can proceed simultaneously with vegetative growth, storage organ formation of garlic plantlets, and D. destructor infection. In 'Fukuchi-white', a variety susceptible to D. destructor, nematodes successfully propagated in storage organs and roots. Furthermore, the nematodes invaded and propagated in the newly formed storage organs. By contrast, 'Kirishima', 'Hirado', and 'Shishimaru' substantially suppressed more the propagation of the nematodes in storage organs and roots than 'Fukuchi-white'. Additionally, the propagation of nematodes in newly formed storage organs was inhibited in these three varieties. 'Shishimaru' showed unique responses to D. destructor infection: nematode propagation was the lowest among six varieties in inoculation tests and the nematode-inoculated cloves turned brown. Our results suggest that several garlic varieties have resistance mechanisms that suppress the propagation of D. destructor in storage organs and roots, and that in vitro inoculation methods are useful for selecting resistant garlic varieties. These findings will help developing novel D. destructor-resistant garlic varieties and our further understanding of garlic-nematode interactions.

Regulation of Different Lights on Energy Acquisitions, Microtuber Formation, and Growth of In Vitro-Grown Solanum tuberosum L.

Limited research has been conducted on the regulation of light quality on heterotrophy in in vitro-grown potato plantlets. Here, we investigated the effect of light quality on photosynthetic and heterotrophic abilities as well as microtuber formation and growth of potato plantlets (Solanum tuberosum L. cv. Shepody). Potato plantlets pre-cultivated under white light for 30 days were then transferred to grow under blue (B), green (G), red (R), yellow (Y), and white (W) lights, and parameters including dry weight, photosynthetic pigment, medium solute consumption, δ13C value, root activity, and sucrose transport (SUT) gene expression of these plantlets were measured. The results showed that B, G, and W were conducive to the rapid induction of microtubers, while R, and especially Y, delayed microtuber formation. Higher photosynthetic ability was observed in the W treatment, whereas the opposite effect was seen in the monochromatic light treatments. Microtuber growth was primarily dependent on heterotrophy, and B was conducive to microtuber growth. The delay in microtuber formation was related to the high expression of StSUT4 in the root, and better microtuber growth was associated with higher root activity, more medium solute consumption, and a higher expression level of StSUT1 in the roots.

Effect of Gamma Irradiation on Embryogenic Calli of Rice and Subsequent Shoot and Regeneration

This study was carried out during May 2021 to January 2022 at the Tissue Culture Laboratory of Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh, Bangladesh for callus induction and regeneration ability of plantlet from low doses irradiated embryogenic calli of Binadhan-13 and BR5 (Dulabhog). The effect of low gamma rays such as 4, 6 and 8 Gy were observed in vitro shoot, root and plantlet formation. Two different incubation conditions such as light and dark incubation were maintained. Between two varieties, Binadhan-13 showed the highest ability of callus induction in both conditions. Callus induction was significantly higher under dark condition (77.14%) than light condition (67.14%) ‍at 21 days. Data revealed that gamma rays affect both shoot and root regeneration ability of embryogenic callus. Shoot regeneration ability was the highest at 4 Gy in Binadhan-13 (60%) and gradually decreased with the increased doses of gamma rays. Among the treatments, shoot regeneration ability (60% in Binadhan-13 and 50% in BR5) was higher at 4 Gy dose of gamma ray followed by 6 Gy where shoot regeneration ability was 55% and 45% in Binadhan-13 and BR5 respectively. In root induction, the highest root induction ability (80%) was observed in the cultivar Binadhan-13 at control condition. Like shoot regeneration, 4 Gy gamma ray showed better root induction in both the varieties (70% in Binadhan-13, 60% in BR5). The in vitro regenerated plantlets from irradiated embryogenic calli were successfully transferred to soil in pots. The regeneration protocol could be further used for varietal improvement of rice varieties using nuclear technique (60Co gamma ray). Bangladesh J. Nuclear Agric, 37(2): 29-42, 2023

The Effect of BAP Concentration on In-Vitro Mutant Taro Regeneration

Plant regeneration through invitro culture is strongly influenced by the use of growth regulators. BAP is a class of cytokinins that affect cell division which is very well used to stimulate plant growth. The aim of the study was to determine the effect of BAP concentration on the regeneration of various in vitro mutant taro. The study was carried out at the Plant Tissue Culture Laboratory, Faculty of Agriculture, Hasanuddin University, Makassar. The method used was factorial completely randomized design with 2 factors as the environmental design. The first factor was the BAP concentrations of 0.0 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L and 2.5 mg/L. The second factor was mutant plants consisting of control plants (diploid), mutant 1 ( Putative-Tetraploid), mutant 2 (Mixoploid), mutant 3 (Mixoploid), and mutant 4 ( Putative-Tetraploid ). The results showed that the best regeneration was obtained by BAP concentration of 0 mg/L with control plants on the parameters of root length (2.41 cm), number of roots (11.66 pieces) and plantlet formation rate (13 days). The BAP concentration of 1 mg/L was shown by mutant plant 1 on the parameters of the number of shoots (26 pieces) and number of leaves (16 pieces). BAP concentration of 0 mg/L in mutant 4 had a shoot height of 1.83 cm. The results of ploidy flow cytometry analysis of mutant plants with Putative-Tetraploid parental traits after regeneration resulted in detectable diploid chromosomes that were different from the parent types in all BAP concentration treatments

Somatic embryo formation, plantlet vigorous and greenhouse preliminary test of garlic bulblet obtained through somatic embryogenesis

Garlic production in Indonesia is still low due to limited availability of seedling. Approximately 10% of garlic yield are used for the next cultivation. Therefore about 90 % of garlic consumption are still imported from China. In addition to conventional seed production, alternative routes are needed, such as through somatic embryogenesis. Research consists of three activities, namely 1) induction and multiplication of callus, 2) embryo somatic and plantlet formation and 3) preliminary test of bulblet in vitro in the greenhouse. Parameters observed in the laboratory activities were time of callus emergence, callus weight and morphology, time of somatic embryo emergence, embryo diameter and weight per bottle, plantlet number and vigorous and bulblet number per bottle, while in the greenhouse, parameter observed were survival of bulblet number, plant height, leaf number per plant, and clove number and weight. The result showed that application of 2,4-D 3 mg/l with casein hydrolysate acid was suitable for callus induction of garlic var. Lumbu Kuning, while BAP 2 mg/l was the most suitable formula for callus multiplication, the addition of paclobutrazol 1 ppm and sucrose 20% in MS medium was the most suitable treatment for plantlet vigorous, while the bulblet formation could be developed by using jasmonic acid with sucrose of 12%. The best medium for growjng media of in vitro bulblet in the greenhouse was the combination of sandy soil, cocopeat and manure.

In vitro multiplication of “Kepok” banana (Musa acuminata x Musa balbisiana) using different concentration of BAP and NAA in MS media

This study aims to determine the effect of different composition of BAP and NAA concentration added to Murashige and Skoog (MS) basic media on the in vitro multiplication of Indonesian “Kepok” banana variety. The research was conducted at the Tissue Culture Laboratory of Bonto-Bonto Horticultural Plant Seed Center. A completely randomized design (CRD) was employed with two factors tested. Four concentrations of each BAP and NAA used were 0 ppm, 4.5 ppm, 5 ppm, and 5.5 ppm for BAP, and 0 ppm, 2 ppm, 2.5 ppm, and 3 ppm for NAA resulted in 15 composition of BAP:NAA in addition to control (without the hormones). The results show no interaction between the BAP and NAA treatments, BAP concentration of 5 ppm gave the best result on parameter time to shoots emergence (7.50 DAP). NAA concentration of 2.5 ppm gave the best results on root emergence time (8.61 DAP), time to plantlet formation (11.72 DAP), number of roots (4.81 roots) and root length (5.93 cm).

In vitro somatic embryogenis and plant regeneration from cell suspension culture of endangered plant ‘Virali’ Dodonaea viscosa

An effective protocol was developed for in vitro propagation of ‘Virali’ via somatic embryogenesis in cell suspension culture. Embryogenic callus was obtained on Murashige and Skoog (MS) medium by a relatively moderate concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (4 mg/L), high concentrations of proline and glutamine (both 300 mg/L) and coconut water to develop induction of calli from explant. A suspension culture of the callus-derived embryogenic cells in Murashige and Skoog (MS) basal medium (pH 5.3) with 2 mg/L 2,4-D, 1 mg/L indole-3-acetic acid (IAA), 1 mg/L NAA, 45 g/L sucrose, and 20 g/L maltose resulted in synchronously proliferating cells with the capacity to be induced into somatic embryos on an 8g/L agarose, 20 g/L maltose, 10 g/L dextrose, 40 g/L sucrose, 1 mg/L naphthaleneacetic acid (NAA), and 0.2 mg/L. On an 8 g/L agarose-solidified MS basal medium (pH 5.7) with 1 mg/L NAA, 100 ml/L coconut water, 30 g/L sucrose, 30 g/L maltose, and 100 mg/L glutamine, the young somatic embryos differentiated into regenerable mature somatic embryos. In a 2.5 g/L gelrite solidified MS-based medium with 2.5 mg/L 6-benzylaminopurine (BAP), 1 mg/L gibberellic acid (GA3), 100 mg/L L-glutamine, 30 g/L sucrose, and 2.5 mg thidiazuron, the mature somatic embryos regenerated into plantlets (TDZ). For the formation of plantlets with a height of 10 to 12 cm that could endure the hardening process, it took a total of 6 months from the explant stage. The plantlets’ regular morphology suggests that there was no somaclonal variation in their development. The current regeneration approach for “Virali” has a significant deal of potential for micro propagating endangered germplasm and improving it through transgenic technology.

Increased Zygote-Derived Plantlet Formation through In Vitro Rescue of Immature Embryos of Highly Apomictic Opuntia ficus-indica (Cactaceae).

O. ficus-indica (prickly pear cactus) is an important forage and food source in arid and semiarid ecosystems and is the most important cactus species in cultivation globally. The high degree of apomixis in the species is a hindrance in plant breeding programs where genetic segregation is sought for the selection of superior genotypes. To understand if in ovulo embryo rescue could increase the proportion of zygotic seedlings, we compared the mature seed-derived seedlings with those regenerated from in vitro embryo rescue at 20, 25, 30, 35, and 40 post-anthesis days (PADs) in four Italian cultivars. The seedlings were classified as apomictic or zygotic based on molecular marker analysis using inter-sequence single repeat (ISSR) primers. Multiple embryos were recovered from all the cultured immature ovules, and plantlets were regenerated and acclimatized to the field post hardening, with success rates ranging from 62% ('Senza spine') to 83% ('Gialla'). The level of polyembryony differed among cultivars and recovery dates, with the highest being 'Rossa', producing 4.8 embryos/ovule at 35 PADs, and 'Gialla', the lowest, with 2.7 at 40 PADs. The maximum number of embryos observed within a single ovule was 14 in 'Trunzara bianca'. ISSR analysis revealed that ovule culture at 35 PADs produced the highest percentage of zygotic seedlings in all the cultivars, from 51% ('Rossa') to 98% ('Gialla'), with a high genotype effect as well. Mature seeds produced much fewer seedlings per seed, ranging from 1.2 in 'Trunzara bianca' to 2.0 in 'Rossa' and a lower percentage of zygotic seedlings (from 14% in 'Rossa' to 63% in 'Gialla'). Our research opens a pathway to increase the availability of zygotic seedlings in O. ficus-indica breeding programs through in ovulo embryo culture.

Colchicine Effect of Mitosis Root Tip by Banana Kepok Yellow (Musa paradisiaca L.) Polyploid Explants

Colchicine can inhibit spindle formation so that doubled chromosomes fail to separate during anaphase cytologically to induce mutations that produce polyploid plants. Yellow kepok banana is a triploid banana. The supply of yellow kepok bananas is hampered by conventional cultivation methods and the limited availability of quality seeds. This study aims to determine the effect of adding colchicine compound to tissue culture media on the mitotic index, chromosome number, and chromosomal abnormalities in yellow kepok bananas and the formation of polyploid plantlets. This study was included in the experimental study by conducting mitotic preparation and observing morphology after the test sample was induced by colchicine. The results of the research that has been done show that the addition of the compound colchicine to the tissue culture medium of yellow kepok banana causes a decrease in the mitotic index, an increase in the number of chromosomes, the presence of chromosomal abnormalities, the formation of polyploid plantlets, and an increase in the number of roots, but there is a reduction in root length.

Cryopreservation of Zygotic Embryos of Podophyllum Hexandrum Royle, An Endangered Medicinal Plant, by Vitrification and V Cryo-Plate Techniques

Background: Podophyllum hexandrum is a highly endangered valuable medicinal plant of the belonging to family Berberidaceae. This plant needs conservation efforts due to the over- and unscrupulous harvesting from the wild because of its ever-increasing demand. To establish a long-term cryopreservation method for Podophyllum hexandrum using techniques: Vitrification and V Cryo-plate. MATERIALS AND METHODS: Zygotic embryos cryopreserved using vitrification and V cryo-plate by optimization of parameters including time, loading time and PVS2 dehydration time. Recovery of zygotic embryos was on different regrowth media for plantlet formation. RESULTS: With V cryo-plate, 90% was obtained as compared to 73.3% with vitrification. V Cryo-plate conditions were pre- of zygotic embryos in 0.3 M sucrose for 4 days, treatment in loading solution with 0.8 M for 20 min, dehydration in PVS2 for 50 min, LN exposure, unloading in 1.2 M sucrose for 20 and transfer of zygotic embryos to regrowth medium for recovery. During recovery, the maximum of shoots (4.2) and highest shoot length (5.1 cm) were observed on regrowth medium with 1.5 BAP and 0.1 mg/l IAA (R 7). CONCLUSION: Zygotic embryos of Podophyllum hexandrum with 90% regrowth using a V cryoplate technique and plantlets were produced cryopreservation.

A comparative macromorphological and ethnomedicinal analysis of five Kalanchoe Adans. Taxa from Bangladesh

The best-known species of the succulents family Crassulaceae in Bangladesh are Kalanchoe blossfeldiana, Kalanchoe daigremonitnum, Kalanchoe heterophylla, Kalanchoe laciniata and Kalanchoe pinnata during the period January, 2019 to March, 2020. But this is the first time that two new species named K. delagoensis and K. gastonis-bonnieri were reported from Bangladesh in this study.Viviparous plantlets formation along the leaf margin is the remarkable feature of these new two species along with formerly reported species K. pinnata. The abundance of K. pinnata among the five species of this genus was the highest (53%), while K. gastonis-bonnieri was the lowest (3%). The experimented five species were used to treat 9 human ailments named cough and cold, cuts and wounds, stomach disorder, kidney and gall bladder stones, jaundice, high blood pressure, skin burning, headache, and eye pain. The citation frequency of all the species except K. laciniata ranged from 2% to 100% in contrast to any ailment, while K. blossfeldiana and K. pinnata were frequently cited. Alike citation frequency the species K. blossfeldiana and K. pinnata along K. gastonis-bonnieri had the highest fidelity level, but K. delagoensis had the lowest fidelity level among the five species analyzed. Leaf juice and baked leaf are the common modes of administration. The most cited and high-fidelity species K. blossfeldiana and K. pinnata were top-ranked in DMR analysis. These two top-ranked species are under threat for several factors and need conservation strategies. Bangladesh J. Agri. 2023, 48(1): 51-66

Meristem genes are essential for the vegetative reproduction of Kalanchoë pinnata.

Several Kalanchoë species reproduce asexually by forming plantlets in the leaf crenulations. Some species produce plantlets incessantly via somatic embryogenesis and organogenesis, whereas others exclusively develop plantlets after leaf detachment, presumably through organogenesis. SHOOT MERISTEMLESS (STM), which mediates SAM functions, appears to be involved in Kalanchoë plantlet formation, suggesting that meristem genes may be essential for plantlet formation. However, the genetic regulatory network for establishing and maintaining plantlet primordia in Kalanchoë remains elusive. Here, we showed that meristem genes were differentially expressed in the leaf crenulations of K. pinnata during plantlet development after leaf detachment. The regulatory interactions among these meristem genes are largely conserved in K. pinnata crenulations. Moreover, transgenic antisense (AS) plants with lower expression of these key meristem genes formed significantly fewer plantlets with some morphological defects, suggesting that the meristem genes play an important role in plantlet formation and development. Our research revealed that key meristem genetic pathways were co-opted to the leaf margin to facilitate the unique asexual reproduction mechanism in K. pinnata. This also highlights how evolutionary tinkering invents new structures such as epiphyllous buds and plantlets by rewiring pre-existing genetic pathways.

Indirect somatic embryogenesis in sweet orange cv. “Mosambi”

This study was carried out for standardization of a reliable protocol by using different explants (epicotyl,cotyledon, and root), plant bioregulators (PBRs), and carbon sources on indirect embryogenesis during 2019-2022. Among the treatments tested, T4 (MS + 2,4-D (1.5 mg L-1) + BAP (1.0 mg L-1) + ME (500 mg L-1)) proved bestfor callusing (90.89 %), callus fresh weight (0.83 g), turgid weight (0.84 g) and dry weight (0.08 g) as comparedto others. In terms of callusing (83.72%), callus fresh weight (0.67 g), turgid weight (0.68 g), dry weight (0.05g) and callus water content (11.28 %), explants epicotyl (E) showed its superiority over others. Similarly, thetreatment combination T4 × E surpassed others in callusing potential. Embryogenesis (59.09%) and germination(33.61%) were best in T7 (MS + BAP (2 mg L-1) + NAA (0.1 mg L-1) + ME (500 mg L-1)). Among the different carbonsources, 5% glycerol supplemented with T7 proved best in inducing the highest number of somatic embryos/callus mass (73.26), embryogenesis (65.27%), and plantlet formation (68.77%). The standardized protocol canbe used for indirect embryogenesis for different genotypes of sweet orange.

Efficient Somatic Embryogenesis, Regeneration and Acclimatization of Panax ginseng Meyer: True-to-Type Conformity of Plantlets as Confirmed by ISSR Analysis

Panax ginseng Meyer grows in east Russia and Asia. There is a high demand for this crop due to its medicinal properties. However, its low reproductive efficiency has been a hindrance to the crop's widespread use. This study aims to establish an efficient regeneration and acclimatization system for the crop. The type of basal media and strength were evaluated for their effects on somatic embryogenesis, germination, and regeneration. The highest rate of somatic embryogenesis was achieved for the basal media MS, N6, and GD, with the optimal nitrogen content (≥35 mM) and NH4+/NO3- ratio (1:2 or 1:4). The full-strength MS medium was the best one for somatic embryo induction. However, the diluted MS medium had a more positive effect on embryo maturation. Additionally, the basal media affected shooting, rooting, and plantlet formation. The germination medium containing 1/2 MS facilitated good shoot development; however, the medium with 1/2 SH yielded outstanding root development. In vitro-grown roots were successfully transferred to soil, and they exhibited a high survival rate (86.3%). Finally, the ISSR marker analysis demonstrated that the regenerated plants were not different from the control. The obtained results provide valuable information for a more efficient micropropagation of various P. ginseng cultivars.

Sprout formation in epiphyllous plantlets from detached Bryophyllum calycinum leaves: Relevance for the senescence of mother leaves under drought stress

Epiphyllous plantlets / vegetative propagules of Bryophyllum calycinum have been well known to develop only on detached leaves but not in leaves of intact plants. In the present study we have found a morphogenesis that the intact epiphyllous plantlets induce sprouts, but not epiphyllous buds, in plantlet leaves when the mother leaves almost completely senesced under drought stress. Anatomical studies of sprouts showed the presence of vascular connection between sprouts and plantlet leaves. After re-watering isolated plantlets, the growth of existing sprouts was inhibited and most of the primordia of buds in plantlet leaves were little differentiated, they appeared to be dormant and further formation of plantlets was suppressed. The sprouts were not formed when plantlets were connected with green parts of mother leaves. Removal of primordia on the one side of detached mother leaves caused loss of chlorophyll in leaves, and the appearance of plantlets on the other side delayed chlorophyll degradation. These results suggest that development of sprouts from epiphyllous plantlets on detached mother leaves is closely related to leaf senescence induced by severe drought stress. The role of the total senescence of the mother leaves under drought conditions in the induction of sprouts is discussed with respect to hormonal balance and signaling networks.

Influences of Organic Additives on Asymbiotic Seed Germination of Dendrobium cruentum Rchb. f. for In Vitro Micropropagation

Dendrobium cruentum Rchb. f., a commercial orchid in Thailand, faces the risk of extinction and should be conserved, and propagated for commercial. Therefore, the aim of this research was to identify a suitable VW medium supplemented with organic additives for the micropropagation of D. cruentum through asymbiotic seed germination. The VW media supplemented with separate organic additives (coconut water (CW) (100 mL/L), ‘Hom Thong’ banana homogenate (BH) (1000 mg/L), and potato homogenate (PH) (1000 mg/L) or its combination was determined, and the seed germination rate (%), protocorm formation, and plantlet development were observed. The results showed that a combination of BH and PH resulted in the highest seed germination (94.69 ± 1.55 %) when compared to basal VW (control). Protocorm formation was highest with CW alone (47.38 ± 7.17 %) and a combination of CW and PH (52.13 ± 6.20 %). Additionally, after 10 weeks of culture, supplementation with BH yielded the highest number of plantlets. However, after 24 weeks of culture, each organic additives gave the different developments of healthy plantlets. To investigate suitable potting media for acclimatization of well-grown plantlets in greenhouse conditions, 2 pieces of foam, dried coconut husk, and a combination of peat moss: Sphagnum moss: Pumice: Perlite (3:1:1:1) were investigated. The highest survival rate percentage was found in foam-planted plantlets (80 %). Ultimately, the effectively simple mediums containing organic additives and a one-step protocol were provided for the micropropagation of D. cruentum via seeds, and the derived information could be used for D. cruentum conservation and production. HIGHLIGHTS The effects of organic additives on asymbiotic seed germination for in vitro micropropagation of Dendrobium cruentum Rchb. f. was firstly reported The media supplemented with organic additives yield significantly the highest seed germination, protocorm formation, and plantlet formation and development of D. cruentum when compared to basal media In greenhouse conditions, 2 pieces of foam gave the highest survival rate percentage of plantlets (80 %) The practical and beneficial media supplemented with organic additives for micropropagation of D. cruentum was provided GRAPHICAL ABSTRACT

Biochars as culture medium additives influence organogenic potential of plant explants through changes in endogenous phytohormone and carbohydrate contents in Daphne species

Biochar applicability as culture medium amendment is rarely investigated due to its uncovered impact on in vitro cultured plants. This study assessed the feasibility of several biochars as alternatives to activated charcoal, for micropropagation of two ornamental Daphne species (Thymelaeaceae). We distinguished metabolic responses that were specific for: a) medium supplementation with any type of charcoal; b) biochar addition; c) biochar activation; d) the process of charcoal activation itself. We compared impacts of industrially produced biochar and three different biochars made in small-scale stoves (from residues of Gliricidia sepium) on organogenic capacity and physiological status in Daphne sp. shoots. Metabolite profiling revealed that biochars differently modulated accumulation of endogenous phytohormones and osmolytes in the shoots. Biochars reduced accumulation of proline, betaines and stress-related phytohormones (ABA, jasmonates), and impacted carbohydrate profile. For D. jasminea the most impactful was biochar activation, which increased micropropagation efficiency by elevating cytokinin and soluble sugars content. For D. tangutica charcoal/biochar activation, and application of biochar reduced accumulation of ABA and jasmonic acid, increased level of gibberellins, salicylic and benzoic acid, and altered carbohydrate composition. The study revealed that tested biochars can be used as a sustainable medium supplements without negative impact on production of new microshoots. Based on studied species we showed that biochar supplements could stimulate plantlet formation (D. jasminea,) ameliorate stress response (D. tangutica), and constitute a way of undesired biomass utilization (invasive species G. sepium).

Evaluation of chemical components of rhizomes and micropropagation from Kaempferia rotunda L.

In this study, the extracts of Kaempferia rotunda L. were investigated by GC/MS (Gas chromatography/mass spectrometry). There were 25 compounds in tuberous rhizome: alpha-pinene (4.48%), camphene (20.85%), pentadecane (15.47%), camphor (10.15%), alpha terpinolene (1.01%), bornyl acetate (5.65%), alpha-selinene (2.32%), gamma-curcumene (3.22%), heptadecane (3.80%), alpha-cedrene (3.64%), alpha-amorphene (4.92%), alpha-curcumine (2.68%), benzyl-benzoate (7.56%), eucalyptol (1.01%), and some important other compounds. The protocol for in vitro propagation was conducted. The MS medium supplemented with 2 mg/L of Benzyl adenine and 0.2 mg/L of Kinetin gave the highest number of shoots (4.67 shoots/explant). The MS 1/2 medium supplemented with 0.5 mg/L of Naphthalene acetic acid was suitable for plantlet formation (shoot height: 12.05 cm and 14.56 roots/explant). The greenhouse-acclimated in vitro plants reached a 100% survival rate.

Comparative Transcriptome Analysis of Two Kalanchoë Species during Plantlet Formation.

Few species in the Kalanchoë genus form plantlets on their leaf margins as an asexual reproduction strategy. The limited molecular studies on plantlet formation show that an organogenesis ortholog, SHOOTMERISTEMLESS (STM) and embryogenesis genes, such as LEAFY COTYLEDON1 (LEC1) and FUSCA3 are recruited during plantlet formation. To understand the mechanisms of two Kalanchoë plantlet-forming species with different modes of plantlet formation, RNA-sequencing analysis was performed. Differentially expressed genes between the developmental stages were clustered in K. daigremontiana (Raym.-Hamet and H. Perrier) and K. pinnata (Lam. Pers.), respectively. Of these gene clusters, GO terms that may be involved in plantlet formation of both species, such as signaling, response to wounding, reproduction, regulation of hormone level, and response to karrikin were overrepresented. Compared with the common GO terms, there were more unique GO terms overrepresented during the plantlet formation of each species. A more in-depth investigation is required to understand how these pathways are participating in plantlet formation. Nonetheless, this transcriptome analysis is presented as a reliable basis for future studies on plantlet formation and development in two Kalanchoë plantlet-forming species.

Improving Agarwood (Aquilaria malaccensis Lamk.) Plantlet Formation Using Various Types and Concentrations of Auxins

<p><em><span lang="EN-US">Aquilaria malaccensis</span></em><span lang="EN-US"> Lamk</span><span lang="EN-US">.</span><span lang="EN-US"> is one of the most </span><span lang="EN-US">widespread</span><span lang="EN-US"> agarwood-producing plants </span><span lang="EN-US">that</span><span lang="EN-US"> face extinction due to overexploitation. Agarwood propagation using <em>in vitro </em>culture techniques is capable of producing large quantities of plants in a shorter time and free from pests and diseases. </span><span lang="EN-US">Therefore, this </span><span lang="EN-US">study</span><span lang="EN-US"> was conducted to analyze</span><span lang="EN-US"> the effect of auxins type and concentration on agarwood plantlet formation using a split-plot design. The main plot was the type of auxin which included IAA, IBA and NAA</span><span lang="EN-US">, while the</span><span lang="EN-US"> subplot was the concentration used which consisted of 0; 5; 10; 15 and 20 µM. The variable observed was agarwood plantlet formation with parameters measured including the number of shoots</span><span lang="EN-US"> and</span><span lang="EN-US"> leaves, plant height, and number of roots. The results showed that the formation of agarwood plantlets was controlled by the type, concentration, and interaction between the type and concentration of auxin. Furthermore, explants cultured on Murashige Skoog (MS) medium supplemented with 10 µM IBA produced the highest number of shoots (3.39 shoots explant<sup>-1</sup>) and leaves (7.25 leaves explants<sup>-1</sup>), while the addition of 10 uM NAA resulted in the highest number of roots (2.52 roots explant<sup>-1</sup>). This is the first time a study is conducted to evaluate the effect of type and concentration of auxins on agarwood plantlet formation</span><span lang="EN-US">.</span><span lang="EN-US"> The production of high-quality shoots and plantlets increased agarwood germplasm availability to prevent extinction and support sustainable production.</span></p>