Callus Induction Medium Research Articles

Efektifitas Madu Sebagai Substituen Media Induksi Kalus Sorgum (Sorghum bicolor) Secara In Vitro

Sorghum is one of the five most important cereal crops in the world, due to its multi-beneficial usages and wide adaptability, so it has the high potential to be developed. One of the current efforts to develop sorghum is through a modern technique, molecular-based. Thus, in vitro culture is an indispensable basic technique, especially in the callus formation process. In addition to commercial synthetic chemicals, various organic materials found in nature have the potential to be used as PGR in initiating callus formation, one of which is honey. This study aims to obtain the optimum concentration of honey as a substituent of sorghum callus induction medium. The research design used a completely randomized design with one factor (honey concentration), which consisted of 5 dffrent levels (0, 5, 15, 25 and 35 gL-1). Observation variables consisted of callus formed (hsi), percentage of callus formation (%) and callus morphology. Data were analyzed using the F variance test and DMRT test at the 5% level. The test results of this study indicate that the two variables are significantly different. The fastest callus formation was in the M0 medium (0 gL-1.), while in the percentage of callus formation. The best results were in the M4 honey treatment (35 gL-1) of 77.78%. Thus it can be seen that the administration of honey as a substituent of in vitro culture media can help increase the success of sorghum callus formation.

Enhancement of shoot regeneration by treatment with inhibitors of auxin biosynthesis and transport during callus induction in tissue culture of Arabidopsis thaliana.

In two-step culture systems for efficient shoot regeneration, explants are first cultured on auxin-rich callus-inducing medium (CIM), where cells are activated to proliferate and form calli containing root-apical meristem (RAM)-type stem cells and stem cell niche, and then cultured on cytokinin-rich shoot-inducing medium (SIM), where stem cells and stem cell niche of the shoot apical meristem (SAM) are established eventually leading to shoot regeneration. In the present study, we examined the effects of inhibitors of auxin biosynthesis and polar transport in the two-step shoot regeneration culture of Arabidopsis and found that, when they were applied during CIM culture, although callus growth was repressed, shoot regeneration in the subsequent SIM culture was significantly increased. The regeneration-stimulating effect of the auxin biosynthesis inhibitor was not linked with the reduction in the endogenous indole-3-acetic acid (IAA) level. Expression of the auxin-responsive reporter indicated that auxin response was more uniform and even stronger in the explants cultured on CIM with the inhibitors than in the control explants. These results suggested that the shoot regeneration competence of calli was enhanced somehow by the perturbation of the endogenous auxin dynamics, which we discuss in terms of the transformability between RAM and SAM stem cell niches.

Callus Regeneration of Gynura procumbens (Lour.) Merr. In Vitro

Gynura procumbens (Lour.) Merr. is one of the species of Asteraceae which is potential as medicinal. Propagation of the species could be conducted by vegetative, so the plant genetic variability is narrow. Genetic variability could be increased by somaclonal variation through callus culture. There is no report on in vitro regeneration from callus culture, although this method could assist further genetic improvement of plants. In this experiment, different concentrations of 2,4- D (0.1 ; 0.3 ; 0.5 mg L -1 ) singly or combination with BA (0.1 ; 0.3 and 0.5 mg L -1 ) were evaluated for callus induction and several concentrations of BA (0 ; 0.1; 0.5 and 1.0 mg -1 ) combination with kinetin (0.1 and 0.3 mg-1) were observed for ability of callus formed shoots. The results showed that the best media for callus induction was 0.5 mg L -1 2,4-D + 0.5 mg L -1 BA. This treatment produced friable callus structure, no roots and yellowish white. Callus regeneration was obtained on the combination of 0.1 mg L -1 BA. + 0.1 mg L -1 kinetin and 0.1 mg L -1 BA + 0.5 mg L -1 kinetin but the percentage was still low. Keywords: G procumbens {Lour.} Merr, induction, regeneration, calli, in vitro

In vitro culture initiation and regeneration of two highly productive clones of poplar

In vitro shoot regeneration is a fast and reliable method for propagation of valuable tree genotypes and important step in genetic manipulations. This study aimed to establish an optimal in vitro culture initiation and regeneration protocol for highly productive poplar clones ‘Novoberlinska-3’ (Populus pyramidalis × P. laurifolia) and ‘Volosystoplidna’ (P. trichocarpa). In vitro culture was initiated either on MS (IM-MS) or WPM (IM-WPM) medium both containing BAP (0.2 mg.L-1) and IBA (0.1 mg.L-1). Results demonstrated that the best initiation medium for the clone ‘Volosystoplidna’ was IM-WPM on which 93.3 % of explants survived, while explants from ‘Novoberlinska-3’ better survived on IM-MS. After establishing both genotypes into aseptic culture, regeneration experiments were started using two types of explants, leaf and petioles, planted on callus induction medium containing 2iP (1.02 mg.L-1) and NAA (1.86 mg.L-1). Furthermore, microshoots were placed on shoot induction medium supplemented with 0.04 mg.L-1 thidiazuron, and then transferred on shoot elongation medium with 0.2 mg.L-1 BAP. Regeneration protocol showed better performance of poplar ‘Novoberlinska-3’, but it was also efficient for ‘Volosystoplidna’. Plant regeneration from leaf explants in the clone ‘Novoberlinska-3’ showed the highest regeneration percentage (92.3 %) and the number of shoots per explant (3.1), which significantly exceeded other explants. Our results showed a significant difference between the survivability of two clones during culture initiation. The differences in regeneration rates between the clones as well as leaf and petiole explants were also determined. Obtained aseptic cultures of highly productive poplar hybrids will be used in our further studies, especially for genetic transformation.

An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. ‘84K’). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and β-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.

Mutagenic efficiency of gamma rays on in-vitro regeneration in Indian mustard (Brassica juncea)

In the study Indian mustard [Brassica juncea (L.) Czern. & Coss.] was treated with different doses (0, 2.5, 10, 20 and 100 Gy) of gamma rays (radioactive cobalt, 60Co) and the effects were investigated on callus initiation, seed germination, seedling growth, total chlorophyll, protein and proline contents of seedling leaves, which were grown from hypocotyl explant of non-irradiated and irradiated seeds. Experiment was conducted at School of Biotechnology, Gautam Buddha University during 2017–18. The results showed that irradiated seeds had increased seed germination per cent, seedling length, fresh and dry weight, total chlorophyll, protein and proline content in low dose exposure (2.5Gy). Callus cultures were established from hypocotyl explants of 15 days old seedlings obtained from irradiated and non-irradiated seeds of B. juncea on MS1 [MS+BAP (2 mg/ml) + 2,4 D (1 mg/ml)] and MS2 [MS+BAP (2 mg/ml) + 2,4 D (0.5 mg/ml)] medium. Cultured hypocotyls formed callus at their cut ends within a week and simultaneously differentiation started into nodular structures from their base. MS1 callus induction medium gave good callus induction percentage (58%) as compared to MS2. It was observed that the callus was green, compact and showed 83% induction frequency in 100Gy gamma dose in MS1, whereas in different dose treatment (2.5–20Gy), callus was white and friable. It turned light brown in 100Gy treatment in MS2 medium. Thus, results of the present study showed pre-sowing seed irradiation is an effective method of improving seed germination at the early developmental stage of the plant.

Improvement of drought tolerance of patchouli through gamma irradiation and in vitro selection

The research was undertaken to obtain drought-tolerant patchouli putative mutant calli through gamma-irradiation and in-vitro selection using Polyethylene Glycol (PEG). Patchoulina 2 variety was used as the mother plant. Embryogenic calli were induced on three medium formulation (combination of 0.1; 0.3 and 0.5 mg/l 2.4D with 0.1 mg/l BAP). The induced embryogenic calli were then exposed to nine levels of Gamma irradiation (0, 5, 10, 15, 20, 25, 30, 35, and 40 Gray) in combination with two levels of PEG (0 and 20%). The results showed that MS media with 2.4-D 0.3 mg/l and BAP 0.1 mg/l was the best medium for callus induction. The ability of calli to survive decreased with increasing doses of gamma irradiation. The radiosensitivity of patchouli calli showed that LD20 at 14.06 Gray and LD50 at 26.98 Gray. The drought-tolerant patchouli putative mutant calli were obtained from gamma-ray irradiation doses 15, 20, and 25 Gray in selection media 20% PEG has better drought stress tolerance compared to parent Patchoulina 2 variety. Further selection in the glasshouse and field are needed to obtain a candidate variety of patchouli drought tolerance.

Optimization of Somatic Embryo Regeneration of <i>Larix olgensis</i> Through Multiple Media

To establish a stable and efficient  Larix olgensis  somatic embryo regeneration system. In this study, immature zygotic embryos of  Larix olgensis  were used as explant materials for embryogenic callus induction, and embryogenic callus with good growth were selected to induce somatic embryo formation by liquid medium A, semi-solid medium B, and solid medium C. The callus induction medium is S medium+2,4-D 1.0 mg/L + 6-BA 0.5 mg/L + KT 0.3 mg/L, sucrose 20 g/L, agar 3 g/L; the subculture medium is S medium+2, 4-D 0.15 mg/L + 6-BA 0.05 mg/L + KT 0.05 mg/L, sucrose 30 g/L, agar 6 g/L. Mature medium A is S medium + PEG4000 100 g/L + ABA 15 mg/L + AgNO 3  10 mg/L, sucrose 45 g/L liquid medium. Mature medium B and C are mature medium A supplemented with 2 g/L and 10 g/L agar, respectively. The maturation time of this method is about 32 days, the synchronization rate is 78.0%, and the proportion of somatic embryos in normal morphology is 83.0%. Somatic embryo formation efficiency in normal morphology can reach 223.0 embryos/g embryogenic callus, somatic embryo germination rate can reach 47.2%. Compared with a single maturation medium, the maturation time is shortened, somatic embryo generation is more synchronized, and high somatic embryo yield and quality are improved. It provides a way for the commercialization of somatic embryo regeneration of  Larix olgensis , brings great convenience to large-scale breeding and reproduction, physiological and biochemical research, and genetic modification, and has great practical application value.

Establishment of Rapid Propagation System of <i>Lycium Ruthenicum</i> Murr Tissue Culture

Lycium ruthenicum  is a perennial shrub of Lycium in Solanaceae, which is an important plant for improving the environment of desert greening. The sterile seedlings of  Lycium ruthenicum  were inoculated in callus induction medium MS-1 (MS+0.5~1 mg/L NAA+1.5~2.5 mg/L 6-BA+30 g/L sucrose + 8 g/L agar, pH5.2) for about 10 days. After 40 days, the callus was separated from the explants and placed in MS-2 medium (MS + 0.5 ～ 1 mg/L NAA + 0.5~1.4 mg/L 6-BA + 30 g/L sucrose + 6 g/L agar, pH5.2) to induce adventitious buds. Finally, the buds were inoculated in MS medium to take root. Rooting in MS medium about 20 days later, the seedlings grew up, each seedling had 3-4 roots, and then carried out seedling refining and transplanting. This method of callus induction is due to obtaining callus directly from sterile seedlings of  Lycium ruthenicum . And it shorts the time of tissue culture, reduces the cost of culture and improves the efficiency of culture in  Lycium ruthenicum .

Histological studies on somatic embryogenesis in rice (Oryza sativ L.)

Rice (Oryza sativa) embryogenic calli were obtained from mature zygotic embryos culture using two varieties of rice, Sakha 101 and Sakha104 on Murashige and Skoog (1962 ) medium supplemented with 1mg/l, 2mg/l and 3mg/l 2,4- dichlorophenoxy acetic acid. The results indicated that the best medium was that containing 3mg/l. Histological analysis of somatic embryogenesis revealed that after two weeks of culture of explants on the callus induction medium, somatic embryo development began with a cluster of proembryogenic cells in the peripheral region of the calli. The outer cell layer of embryogenic calli consisted of small and isodiametric cells, each with a dense cytoplasm and a prominent nucleus and nucleolus, whereas the inner cell layer is composed of large cells with small nuclei and large vacuoles. These embryogenic cells underwent a series of organized divisions and formed the proembryo with a well-defined protodermis. At 20 days after initiation of culture, these proembryos continued a series of organized divisions and gave rise to globular somatic embryos which are delimited by a well-defined layer of epidermal cells with conspicuous nuclei. These somatic embryos had no apparent vascular connection with the mother tissue and had a suspensor. After ten days of culture on regeneration medium, globular somatic embryos developed into heart-shaped somatic embryos, which consisted of cells with prominent nuclei and dense cytoplasm. After 15 days of culture on regeneration medium torpedo-shaped somatic embryos were observed. Then, after 25 to30 days, it turned into a monocotyledon form.

Establishment of Tissue Culture Regeneration System for Medicago ruthenica L. cv. ‘Zhilixing’

Background: Medicago ruthenica L. ‘Zhilixing’ is a new variety with superior forage and seed yield compared to the wild type. The cold, drought and salt tolerance of Zhlixing are better than those of alfalfa, suggesting that this variety can serve as a high-quality genetic resource for improving the stress resistance of alfalfa. However, because of the lack of tissue culture regeneration system, it is difficult to perform genetic transformation studies on stress resistance genes. This study aimed to establish an efficient tissue culture regeneration system for Zhilixing variety. Methods: Three types of explants were selected and tested on four types of basal media supplemented with different combinations of auxin and cytokinin for callus induction and differentiation, based on orthogonal tests to select the combinations of auxin and cytokinin suitable for callus induction and differentiation. Two-factor combination method was used to formulate a suitable rooting medium. Result: The hypocotyledonary axis was found to be an excellent explant for callus induction on MS medium. The optimum callus induction medium contained thidiazuron (TDZ, 0.5 mg/L), 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg/L) and naphthaleneacetic acid (NAA, 0.5 mg/L) where the callus induction rate was 93.33%. The differentiation medium was supplemented with TDZ (0.75 mg/L), 2,4-D (0.25 mg/L) and 6-benzyladenine (6-BA, 1.5 mg/L) where the differentiation rate was 63.33%. Thidiazuron played the key role in both processes of callus induction and differentiation. Half-strength MS containing 0.1 mg/L of NAA was the most efficient rooting medium.

PROPAGATION OF PITCHER PLANTS (Nepenthes gracilis KORTH. AND Nepenthes reinwardtiana MIQ.) THROUGH CALLUS INDUCTION

In vitro propagation of pitcher plants is still limited only using seeds, while the other in vitro methods using leaf explants such as embryogenesis, organogenesis, and callus culture has not been widely reported. The research aims to study the growth response of leaf explants in two species of pitcher plants (Nepenthes gracilis and Nepenthes reinwardtiana), which formed callus in several treatment media combinations. Leaf pieces were taken from a 4-month pitcher plant culture were grown on the treatment media, namely modified media of Murashige & Skoog with a half concentration (½ MS) added 2.4-Dichlorophenoxoxyacetate (2.4-D) with a concentration of 0, 0.5, 1, 1.5, and 2 mg L-1 and kinetin (0.5 mg L-1). The treatment was then stored in a dark environment to induce callus formation. The observation for 12 weeks showed that the time of callus formation in two species of pitcher plant observed was not significantly different. Both species of pitcher plants begin to form callus in the fourth week after treatment. None of the leaf explants were planted on the control medium without hormones formed callus. The best medium for callus induction in N. gracilis is ½ MS medium added with 2 mg L-1 2,4-D and 0.5 mg L-1 kinetin, with callus morphology brownish-white with friable texture. In comparison, the optimum callus media from leaf explant of N. reinwardtiana has not been obtained yet. Thus further research is still needed.

The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii.

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

Genomic Methylated Cytosine Level during the Dedifferentiation and Cellular Competence in Coffea arabica Lines: Insights about the Different In Vitro Responses

Coffea arabica genotypes present distinct responses in vitro, and somaclonal variation occurrence has been reported. Global cytosine methylation is one of the epigenetic mechanisms that influences the Coffea in vitro responses. We aimed to establish the indirect somatic embryogenesis in C. arabica ‘Catuaí Vermelho’, ‘Caturra’ and ‘Oeiras’, associate the distinct responses to the methylated cytosine genomic level, and check the ploidy stability. Leaf explants were cultured in callus induction and proliferation medium. The resulted calli were transferred to the regeneration medium, and the mature cotyledonary somatic embryos were transferred to the seedling medium. ‘Oeiras’ exhibited the highest number of responsive leaf explants, followed by ‘Caturra’ and ‘Catuaí Vermelho’. Global methylated cytosine level increased over time in the ‘Catuaí Vermelho’ and ‘Caturra’ friable calli, remaining constant in ‘Oeiras’. ‘Oeiras’ did not regenerate somatic embryos, while ‘Catuaí Vermelho’ exhibited the highest number. Somatic embryo regeneration was associated with the increase of the methylated cytosine level. However, the ‘Catuaí Vermelho’ embryogenic calli showed a lower methylated cytosine level than ‘Caturra’. Recovered plantlets exhibited the same 2C value and chromosome number to the explant donors. Therefore, cytosine hypermethylation occurred during C. arabica indirect somatic embryogenesis, influencing cell competence and somatic embryos regeneration.

Pluripotency acquisition in the middle cell layer of callus is required for organ regeneration.

In plant tissue culture, callus forms from detached explants in response to a high-auxin-to-low-cytokinin ratio on callus-inducing medium. Callus is a group of pluripotent cells because it can regenerate either roots or shoots in response to a low level of auxin on root-inducing medium or a high-cytokinin-to-low-auxin ratio on shoot-inducing medium, respectively1. However, our knowledge of the mechanism of pluripotency acquisition during callus formation is limited. On the basis of analyses at the single-cell level, we show that the tissue structure of Arabidopsis thaliana callus on callus-inducing medium is similar to that of the root primordium or root apical meristem, and the middle cell layer with quiescent centre-like transcriptional identity exhibits the ability to regenerate organs. In the middle cell layer, WUSCHEL-RELATED HOMEOBOX5 (WOX5) directly interacts with PLETHORA1 and 2 to promote TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 expression for endogenous auxin production. WOX5 also interacts with the B-type ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and represses A-type ARRs to break the negative feedback loop in cytokinin signalling. Overall, the promotion of auxin production and the enhancement of cytokinin sensitivity are both required for pluripotency acquisition in the middle cell layer of callus for organ regeneration.

English

The genome engineering methods have gained much appreciation for hitting plant disease resistance panorama since the last decade. The in vitro regeneration system is the foundation part of any research related to genetic engineering in plants. Hence, we established a tissue culture regime for efficient in vitro regeneration response of potato cultivar Lady Rosetta tagged with low to no resistance to late blight on foliage and medium to low resistance to late blight on tubers in potato. In this study, five callus induction media and seven regeneration media were investigated to get the best in vitro regeneration response in Lady Rosetta. Based on a statistical analysis of recorded data, the selected combination was CIM2-28 days old calli-RM7. The genetic fidelity of in vitro regenerated plants on a selected combination was assessed using the SSR markers. The wild-type and in vitro regenerated plants showed the same DNA profile. Moreover, a minimal inhibitory dose of basta was optimized for selecting genetically transformed plants in potato, and a 9mg /L basta dose was selected.

Effect of culture media, auxins and genotypes on plantlet regeneration from oil palm (Elaeis guineensis Jacq.) zygotic embryos through somatic embryogenesis

Aim: This study evaluated efficient culture media for the regeneration of elite material through somatic embryogenesis from oil palm zygotic embryos. Methodology: For callus induction, zygotic embryos of four elite genotypes (G1-264T, G2-238DX17P, G3-37DX17P and G4-237T) were cultured on three basal media (Y3, MS and N6) with different auxin 2 mg l-1 (Picloram, 2,4-D and Dicamba) combinations. Subculture was made every month for three passages. It evaluated various callus characters. The embryogenic calli from callus induction media were transferred to the embryo maturation medium and subcultured until the polyembryoids formed. For shoot and root formation, somatic embryo clumps were transferred into regeneration media. In-vitro plantlets with well-grown roots were hardened in pots for six weeks and assessed clonal fidelity using polymorphic SSR primers. Results: Among the treatments, calli from N6+2,4-D, Y3+2,4-D and N6+Picloram showed the highest embryogenic callus potential. G4-237T induced more embryogenic calli (32.982) among genotypes, which was on par with G1-264T (24.196). Embryogenic calli grown on N6 media with Dicamba showed the highest proliferation rate (1.141). After 60 days of culture on regeneration media, the highest number of plantlets per somatic embryogenic clump was obtained from G1-264T on N6 media supplemented with Dicamba. Interpretation: Culture media salt concentration showed a significant difference among media by causing perturbations of auxin flow during somatic embryogenesis affecting callus induction, proliferation and plantlet regeneration. This may be useful for standardizing the genotype-specific regeneration media in oil palm.

Effect of medium composition, genotype and age of explant on the regeneration of hexaploid plants from endosperm culture of tetraploid kiwiberry (Actinidia arguta)

Endosperm, an ephemeral and storage tissue, serves as a source of nutrition and protection during embryo development and germination. It can be used for the cultivation of polyploid plants in vitro. Here, results of plant regeneration and acclimatization from the endosperm-derived calli of four cultivars of Actinidia arguta has been presented. Seeds excised from fresh fruit and dry seeds stored for one year served as the sources of endosperm explants of selected tetraploid cultivars of A. arguta. Callus Induction Medium (CIM; containing 0.25, 0.5, or 1 mg/l of TDZ) and Actinidia Endosperm Medium (AEM; containing 2 mg/l of 2,4-D and 5 mg/l of kinetin) were used to study the organogenic responses of the calli. On AEM, the source of explant did not significantly affect the rate of callus induction for any of the tested cultivars; no organogenic events were observed. In contrast, on CIM both the source of explants and the cultivar origin caused significant differences in callus formation and subsequent organogenic events. Histological and ultrastructural analyses revealed the adventitious nature of shoot bud formation on these media. The most efficient elongation of shoot buds was achieved after transferring organogenic calli with adventitious shoot buds to a medium supplemented with zeatin or meta-topolin. Robust root induction with minimal basal callus formation occurred on the medium with indole-3-acetic acid. Flow cytometric analysis revealed that the nuclear DNA content in the leaves of some regenerants was approximately 50 % higher (4.5 pg/2C) than that in leaves from the tetraploid seedlings (3.1 pg/2C),which confirmed that those regenerants originated from the endosperm. The regeneration of such hexaploid plants was more efficient when endosperm from fresh seeds served as an explant; therefore, fresh rather than dry seeds are recommended for endosperm-derived plant production. The hexaploid plants of A. arguta can serve as an important source of breeding material.

Establishment of callus induction system, histological evaluation and taxifolin production of Larch

Taxifolin in larch is an important natural active ingredient, which prevents and treats liver diseases and cardiovascular diseases, and has many health benefits and application values ​​such as anti-tumor, anti-virus, and anti-aging. However, the traditional extraction method is not conducive to the protection of the ecological environment, and also limits the industrial production of taxifolin from larch. Therefore, the purpose of this research is to establish a larch callus culture system and obtain taxifolin from callus. The shoots of the stem-tips of Larix olgensis were used as explants to be cultured. The light conditions, optimal medium and hormone ratio for callus induction were screened to evaluate the callus induction characteristics of larch and its influence on the accumulation of taxifolin. The results showed that: callus formation started on 28 ~ 51 days, and the best conditions for inducing callus were explored: 12 h/d light culture MS + 2,4-D 2 mg/L + 6-BA 1 mg/L + KT 0.1 mg/L; The callus induction rate was 50-80 %; The hormone combination with the highest accumulation of taxifolin was 6-BA 1 mg/L + NAA 0.1 mg/L, reaching 0.477 . In conclusion, this study revealed a suitable induction condition for larch callus culture and taxifolin accumulation.

In Vitro Production and Exudation of 20-Hydroxymaytenin from Gymnosporia heterophylla (Eckl. and Zeyh.) Loes. Cell Culture.

The metabolite 20-Hydroxymaytenin (20-HM) is a member of the quinone-methide pentacyclic triterpenoids (QMTs) group. This metabolite group is present only in Celastraceae plants, and it has shown various biological activities from antioxidant to anticancer properties. However, most QMTs metabolites including 20-HM cannot be synthesized in a laboratory. Therefore, we optimized a plant tissue culture protocol and examined the potential of Gymnosporia heterophylla (synonym. Maytenus heterophylla) to produce 20-HM in an in vitro experiment. For the first time, we reported the optimum callus induction medium with a high percentage success rate of 82% from the combination of 1 mg/L indole-3-butyric acid and 5 mg/L naphthalene acetic acid. Later, our cell suspension culture cultivated in the optimum medium provided approximately 0.35 mg/g fresh weight of 20-HM. This concentration is roughly 87.5 times higher than a concentration of 20-HM presenting in Elaeodendron croceum (Celastraceae) leaves. In addition, we also found that 20-HM presented in a cultivation medium, suggesting that G. heterophylla cells secreted 20-HM as an exudate in our experiment. Noticeably, 20-HM was missing when Penicillium cf. olsonii occurred in the medium. These findings hint at an antifungal property of 20-HM.