Development of an efficient in vitro callus proliferation protocol for edible wild rhubarb (Rheum ribes L.)

Rheum ribes L. is a perennial wild species. Young shoots and flower bunches are freshly consumed, and root and rhizomes are generally used for medicinal purposes. The aim of the present study was to improve the callus proliferation protocol for R. ribes L. under in vitro conditions. For callus induction, hypocotyl explants taken from 14-day old plantlets germinated in Murashige and Skoog (MS) media were cultured in MS media with 9 plant growth regulator (PGR) combinations containing 6-benzylaminopurine (BAP) (2, 3, and 4 mg/L) + naphthylacetic acid (NAA) (0.1, 0.5, and 1 mg/L). Then, for callus proliferation, 4 PGR combinations containing NAA (0.2 mg/L) + thidiazuron (TDZ) (0.5, 1, 2, and 3 mg) were used in the first set of experiments, and 36 PGR combinations containing BAP (1, 2, 3, and 4 mg/L) + indole-3-butyric acid (IBA) (0.2, 0.5, and 1 mg/L), BAP (1, 2, 3, and 4 mg/L) + NAA (0.2, 0.5, and 1 mg/L), and TDZ (1, 2, 3, and 4 mg/L) + NAA (0.2, 0.5, and 1 mg/L) were used in the second set of experiments. At the end of the second set of experiments, the greatest callus regeneration ratios were obtained due to the combinations including BAP and IBA as well as the low-dose TDZ- (especially 1 mg/L) and NAA- (0.2, 0.5, 1 mg/L) combinations. Regarding callus fresh weights, TDZ + NAA combinations were found to be more successful. The greatest callus fresh weight (12.7 ±0.4 g) was obtained from MS medium supplemented with 2 mg/L TDZ and 0.2 mg/L NAA.

Explant, Medium and Plant Growth Regulator (PGR) Affect Induction and Proliferation of Callus in Abies koreana

Korean fir (Abies koreana E.H. Wilson) is a unique Pinaceae tree species endemic in Korea. In recent years, it is believed that climate change has caused many of them to die. Therefore, it has become extremely important to protect and preserve this tree species. In this study, the possibility of callus induction using different explants, media, and plant growth regulators (PGRs) was studied. After the dormancy period in May 2020, needles and stem segments that grew from the leaf buds as the explants were collected from one-year-old shoots. The explants were disinfected and subsequently transferred to culture media supplemented with different combinations of auxins and cytokinins. These explants were cultured in the dark in a culture room with a 16 h photoperiod, day/night temperature of 24/18 °C, and 80% relative humidity. After 8 weeks, significant differences were observed in the callus induction and proliferation, as affected by the explant type, basic medium, and PGR. The stem segments were more suitable as the explants for callus induction than needles were. Furthermore, fluffy calli suitable for differentiating the regeneration buds were observed on the calli induced from stem segments. The Murashige and Skoog (MS) medium was the most effective of the three media used in this study, namely MS, Douglas fir cotyledon revised (DCR), and Quoirin and Lepoivre (LP) media, with the highest callus induction ratio of stem segments being 100.0%. The highest fresh callus weight was also observed on the MS medium (819.3 mg). Moreover, the PGR combinations of α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (6-BA) consistently exerted a positive influence on callus induction throughout this study. In addition, the advantages of these two kinds of PGR were reflected in callus proliferation. The callus proliferation ratio reached 1,147.6% as compared to the initial fresh weight, with a high concentration of 2,4-D (3.0 mg·L−1). In conclusion, the MS medium was optimal for callus induction on the stem segment explants, and 2,4-D promoted callus induction as well as an increased proliferation ratio of callus in A. koreana.

Callus Induction from Root Fragments of Falcataria moluccana Plantlets

Callus induction of F. moluccana (sengon) was still an obstacle to indirect organogenesis regeneration. The purpose of the study was to determine the callus induction formation from root fragments of F. moluccana plantlets. Primary explants (fragments of roots) were cultivated on MS induction basal media and three concentration combination of PGRs (BAP and NAA): 0.5 ml/l BAP; 0,1 ml/l NAA (single PGR); and combination of 0.5 ml/l BAP + 0.1 ml/l NAA (double PGR). When roots were used as explants, high formation rates of callus (more than 70%) were obtained. Highest formation rates of callus were in NAA added at all clones (12 clones), then BAP added (7 clones) and BAP + NAA added (5 clones). The results indicated that BAP and NAA concentrations used in the media were influence the producing callus and affect the amount of callus produced from roots of sengon. The addition of NAA also gives higher callus proliferation results than the addition of BAP or the addition of a combination of the two hormones. The results indicated that BAP and NAA concentrations used in the media were influence the producing callus and affect the amount of callus produced from roots of sengon.

Identification of miR397a and Its Functional Characterization in Callus Growth and Development by Regulating Its Target in Liriodendron

Callus growth and development, a crucial process in plant propagation, is involved in hormonal balance and abundant gene regulation. MiRNAs are key regulators in the process of cell differentiation and development. MiR397 was identified as participating in plant growth, development, and response to stress, and it was regulated by targeting the LAC gene. The regulatory function of miR397 during callus growth and development was not clear in Liriodendron. In this study, LhmiR397a and its targets were identified, and its regulatory function between LhmiR397a and LhLAC11 was shown using qRT-PCR and transient expression in protoplasts. Furthermore, to clarify the regulatory function of LhmiR397a-LhLAC11, transgenic calli overexpressing LhMIR397a, LhLAC11, and mLhLAC11 were separately obtained by Agrobacterium-mediated transfer. The results showed that overexpressing LhMIR397a might retard callus proliferation, while overexpressing LhLAC11 or mLhLAC11 could promote callus proliferation. Genes associated with the cell cycle had decreased expression when LhMIR397a was overexpressed, while increased expression was observed when LhLAC11 or mLhLAC11 was overexpressed. Additionally, the calli overexpressed with LhMIR397a could generate early cotyledons 21 days after induction, and the somatic embryo induction time was short compared with other genotypes. This study identified LhmiR397a and its targets and provided a functional characterization of LhmiR397a in callus growth and development by regulating its target in Liriodendron.

Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes

Carsono N, Juwendah E, Liberty, Sari S, Damayanti F, Rachmadi M. 2021. Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes. Biodiversitas 22: 2555-2560. The development of callus in the course of transgenic rice avoids the somaclonal variants. To obtain a high number of normal phenotypes and a low number of somaclonal variants requires an appropriate 2,4-D concentration. In this study, we obtained the best callus induction time and a high number of green plant regeneration for three responsive rice genotypes on different 2,4-D concentrations in NB5 medium. The mature seeds of rice embryos were used as explants. A completely randomized factorial design was applied with four levels of 2,4-D concentrations (0, 1, 3, and 5 ppm), two levels of induction time (one and two weeks), and three rice genotypes (cv. Fatmawati, Nipponbare, and Kitaake). The study revealed that there was no interaction effect among genotype, 2,4-D concentration, and callus induction time. Three rice genotypes performed best in callus proliferation and regeneration. One-week callus induction time showed higher callus growth capacity (CGC) as compared to two-week callus induction time. Shoot regeneration capacity (SRC) was independently affected by genotype as well as by callus induction time. The interaction effect between 2,4-D concentration and callus induction time was observed on plant regeneration capacity (PRC). Without the addition of 2,4-D and 1 ppm of 2,4-D, the green plant regeneration capacity (GRC) was comparatively higher. Addition of 2,4-D showed a significant effect, especially at the plant regeneration stage. We found that one-week callus induction was the best treatment for callus proliferation and plant regeneration. We recommend the use of one-week callus induction and 1 ppm of 2,4-D for rice callus proliferation (sub-culture) and subsequent plant regeneration.

Pluronic F-68 Improves Callus Proliferation of Recalcitrant Rice Cultivar via Enhanced Carbon and Nitrogen Metabolism and Nutrients Uptake

Pluronic F-68 (PF-68) is a non-ionic surfactant used in plant tissue culture as a growth additive. Despite its usage as a plant growth enhancer, the mechanism underlying the growth-promoting effects of PF-68 remains largely unknown. Hence, this study was undertaken to elucidate the growth-promoting mechanism of PF-68 using recalcitrant MR 219 callus as a model. Supplementation of 0.04% PF-68 (optimum concentration) was shown to enhance callus proliferation. The treated callus recorded enhanced sugar content, protein content, and glutamate synthase activity as exemplified in the comparative proteome analysis, showing protein abundance involved in carbohydrate metabolism (alpha amylase), protein biosynthesis (ribosomal proteins), and nitrogen metabolism (glutamate synthase), which are crucial to plant growth and development. Moreover, an increase in nutrients uptake was also noted with potassium topping the list, suggesting a vital role of K in governing plant growth. In contrast, 0.10% PF-68 (high concentration) induced stress response in the callus, revealing an increment in phenylalanine ammonia lyase activity, malondialdehyde content, and peroxidase activity, which were consistent with high abundance of phenylalanine ammonia lyase, peroxidase, and peroxiredoxin proteins detected and concomitant with a reduced level of esterase activity. The data highlighted that incorporation of PF-68 at optimum concentration improved callus proliferation of recalcitrant MR 219 through enhanced carbohydrate metabolism, nitrogen metabolism, and nutrient uptake. However, growth-promoting effects of PF-68 are concentration dependent.

PROLIFERATION OF OIL PALM (Elaeis guineensis Jacq.) EMBRYOGENIC CALLUS WITH REPEATED SUBCULTURES IN LIQUID MEDIUM

The availability of high-quality seeds is now a necessity. This is due to a government program to replace oil palm trees in smallholder plantations with high quality seeds. An efficient protocol to produce a large number of embryos is needed. To increase the number of embryogenic callus production, the callus proliferation experiment was carried out through suspension culture. This study aimed to examine the proliferation ability of embryogenic callus from three different oil palm clones, in several repeated subcultures. Liquid MS media added with 1 ppm 2.4-D and 0.1 ppm NAA were used. Embryogenic callus was weighed by 0.1 - 0.2 g, transferred into the liquid media, shaking at 60-80 rpm and 27 ºC for 8 weeks without light. Continues subcultures were repeated up to 7 times. The results showed that the growth rate of embryogenic callus increased in the third and fourth subcultures and then decreased in subsequent subcultures. It also revealed that the entire embryogenic callus from the first subculture up to seventh subculture still has the ability to regenerate into new plants. These results indicate that oil palm embryogenic callus can be proliferated by suspension culture with a limit up to the fourth subculture. Ketersediaan benih kelapa sawit berkualitas saat ini merupakan kebutuhan karena adanya program pemerintah untuk menggantikan tanaman sawit di kebun-kebun petani. Salah satu cara vegetatif yang dapat dilakukan adalah meningkatkan jumlah kalus embriogenik yang dihasilkan melalui pengembangan kultur suspensi. Penelitian ini bertujuan mengkaji kemampuan proliferasi kalus embriogenik dari tiga klon kelapa sawit, pada beberapa kali subkultur yang berulang. Media cair MS dengan penambahan 1 ppm 2,4-D dan 0,1 ppm NAA digunakan untuk memperbanyak 0,1–0,2 g kalus embriogenik, dikocok pada 60-80 rpm dan suhu 27 ºC tanpa cahaya selama 8 minggu. Subkultur berulang dilakukan hingga 7 kali. Hasil percobaan menunjukkan bahwa kemampuan proliferasi kalus dipengaruhi oleh genotip tanaman induk. Rata-rata kalus embriogenik dapat meningkat pada subkultur ke-3 dan ke-4 dan semakin menurun pada subkultur selanjutnya. Kalus embriogenik hasil proliferasi subkultur pertama hingga ke-7 dapat tumbuh menjadi calon tanaman baru. Hasil ini menunjukkan bahwa kalus embriogenik kelapa sawit dapat diperbanyak dengan kultur suspensi pada batas sampai subkultur ke-4.

Optimization of culture conditions for callus proliferation of Curculigo orchioides Gaertn.

Curculigo orchioides Gaertn is a herbaceous plant that has long been used as a tonic in Vietnam with noticeable health benefits. However, the demand for rhizomes of this species could not be met due to their decreasing number in natural habitats. Despite its vulnerability, there are still not enough researches on producing calli of C. orchioides, which is a method having the capacity of creating a large source of cell biomass for bioactive compounds’ extractions. Thus, this study was conducted to figure out the best conditions for C. orchioides’s callus proliferation. Different light regimes, mineral media, and concentrations of some factors like kinetin (KIN), α-naphthaleneacetic acid (NAA), yeast extract (YE), activated charcoal (AC), and silver nitrate (AgNO3) were examined. It is shown by the results that half-strength MS medium (½ MS) given 0.5 mg/L KIN and MS medium supplemented with 0.5 mg/L KIN and put in the dark (0 light hour : 24 dark hours) are the optimal conditions for callus proliferation, with the highest fresh weights (FWs), dry weights (DWs), and growth indices (GIs) of 3.89 g / 0.45 g / 7.78, and 4.10 g / 0.47 g / 8.20, respectively. Additionally, the inhibitory effects of AgNO3, YE, and AC were demonstrated since there was no observed heavy callus in the media containing those factors.