EFFECT OF 2,4-D AND NAA ON SOMATIC EMBRYOGENESIS FROM APICAL PORTION OF GROUNDNUT (ARACHIS HYPOGAEA L.)

Somatic embryogenesis was carried out epicotyl portion of the mature embryo/apical portion. The somatic embryo induction medium containing 2,4-D or NAA (10.0 to 50.0 mg/l). Of the two concentrations tested 2,4-D (30.0mg/l) recorded the highest percentage of response followed by NAA (30.0mg/l). But the highest number of somatic embryo were recorded in 30.0mg/l of 2,4-D followed by NAA. The apical portion of the mature embryo formed direct embryos without any intervention of callus. The maximum percentage of embryogenic cultures were noticed in 30.0mg/l of 2,4-D followed by NAA at 30.0mg/l. for the differentiation of somatic embryos, the embryogenic masses were transferred to medium without any growth regulator. The maximum number of somatic embryos per culture was recorded in 30 mg/l of 2,4-D followed by 30.0 mg/l of NAA. Keywords: Arachis hypogaea L.,Somatic Embryogenesis, 2,4-D and NAA

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.

Somatic Embryogenesis and Plant Regeneration in Viola canescens Wall. Ex. Roxb.: An Endangered Himalayan Herb

Viola canescens Wall. ex. Roxb. is an important but threatened medicinal herb found at 1500–2400 m above mean sea level in the Himalayas. Overexploitation and habitat preference have put the plant under serious threat. Thus, the present study was undertaken to develop an efficient protocol for in vitro propagation via somatic embryogenesis. The results revealed that plant can be regenerated successfully through somatic embryogenesis using leaf derived calli. Regular subculturing of calli on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D)/indole-3-butyric acid (IBA)/kinetin (Kn) and varying combinations of 2,4-D+Kn induced somatic embryogenesis. The maximum average number of somatic embryos (SE) (19.15 ± 2.66) was induced on the medium with 0.15 + 0.05 mg L−1 of 2,4-D and Kn, respectively, and this medium was used as a control. To enhance somatic embryo induction, the control MS medium was supplemented with l-glutamine (200–400 mg L−1) and casein hydrolysate (1–4%). The maximum average number of SE (27.66 ± 2.67) and average mature SE (13.16 ± 3.48) were recorded on the medium having 2 % l-glutamine and 50 mg L−1 casein hydrolysate. The induced SE were asynchronous, so, to foster their maturation, the culture medium (free from growth regulators) was supplemented with abscisic acid (ABA) and silver nitrate (AgNO3). The maximum average number (35.96 ± 3.68) of mature SE was noticed on MS medium supplemented with 1.5 mg L−1 ABA. Mature embryos had two well-developed cotyledons and an elongated hypocotyl root axis. The development of SE into plantlets was significant for embryos matured on the medium with AgNO3 and ABA, with 86.67% and 83.33% conversion on the medium with 0.20 mg L−1 6-benzylaminopurine (BAP). The plantlets thus produced acclimatized in a growth chamber before being transferred to the field, which showed 89.89% survival. The plants were morphologically similar to the mother plant with successful flowering.

INDUKSI KALUS EMBRIOGENIK DAN EMBRIO SOMATIK DARI EKSPLAN DAUN KULIM (Scorodacarpus borneensis Becc.)

Kulim is one of woody plant that have multifunction as wood source and for spice and medicinal. Generative propagation of this plant have trouble because seed use limited. The use of leaf segment through somatic embryogenesis to solve the problem. The objective of this study is to obtain the best treatment to embryogenic callus induction. The modification of basal medium of Murashige and Skoog was used as growth medium. The experiment was conducted in three stages are callus induction, embryogenic callus and somatic embryo induction. The treatment of 2,4-D (3,0 – 12 mg/l) used for callus induction. For embriogenic callus induction used 2,4-D (3,0 – 12,0 mg/l) combined with NAA 0,5 mg/l. The treatment of thidiazuron (0,1 – 0,7 mg/l) used for somatic embryo induction. The result showed that the treatment of 2,4-D 6,0 mg/l is the best for callus induction with compact of texture, green, dry and non embryogenic. The treatment of combination 2,4-D 12.0 mg/l with NAA 0.5 mg/l is the best for friable callus induction. The treatment of 2,4-D 6.0 mg/l combined with NAA 0,5 mg/l is the best for embryogenic callus induction with very friable of texture, easy to separate, dry, smooth and glossy. Thidiazuron of 0,1 mg/l treatment is the best for somatic embryos induction with the average number of 7,8 somatic embryos.

Improvement of a Genetic Transformation System and Preliminary Study on the Function of LpABCB21 and LpPILS7 Based on Somatic Embryogenesis in Lilium pumilum DC. Fisch

Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L.pumilum system was improved, and the genetic transformation efficiency increased by 5.7–13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.

Full-Length Transcriptome Analysis of the ABCB, PIN/PIN-LIKES, and AUX/LAX Families Involved in Somatic Embryogenesis of Lilium pumilum DC. Fisch.

Plant cell totipotency is one of the 25 major topics in current scientific research, and somatic embryos are good experimental material for studying cell totipotency. Polar auxin transport plays an important regulatory role in somatic embryogenesis (SE). However, little is known about the auxin transport genes and their regulatory mechanisms in Lilium SE. In this study, we applied single-molecule real-time (SMRT) sequencing to Lilium pumilum DC. Fisch. for the first time and obtained a total of 119,649 transcripts, of which 14 encoded auxin transport genes. Correlation analyses between somatic embryo induction and gene expression under different treatments revealed that auxin transport genes, especially ATP-binding cassette (ABC) transporter B family member 21 (ABCB21) and PIN-FORMED (PIN) LIKES 7 (PILS7), may be key players in SE, and the necessary duration of picloram (PIC) treatment to induce SE is as short as 3 days. Our research provides valuable genetic information on Lilium pumilum, elucidating the candidate auxin transport genes involved in SE and their influencing factors. This study lays a foundation for elucidating the regulatory mechanism of auxin transport in SE.

Induksi dan Proliferasi Embriogenesis Somatik In Vitro pada Lima Genotipe Kedelai

ABSTRACT<br /><br />Somatic embryo induction medium was reported to be genotype dependent for soybean. This study was aimed to obtain the optimum medium for embryo somatic induction and proliferation, and to regenerate somatic embryo of five soybean genotypes. Five soybean genotypes (Tanggamus, Anjasmoro, Yellow Biloxi, CG-22-10, and SP-10-4) were used in this study. The research was divided into four steps: (1) embryogenic callus induction of five soybean genotypes, (2) embryogenic callus proliferation of five soybean genotypes, (3) optimation of embryo somatic induction on five soybean genotypes and (4) embryo somatic regeneration of five soybean genotypes. The induction experiment showed that based on number of embryogenic callus, the best somatic embryo-induction medium was 3% sucrose+ NAA 5 mg L-1+2,4-D 5 mg L-1+ Vitamin B5. Embryogenic callus number for each genotype tested was increased on proliferation media of 3% sukrosa + 2,4-D 5 mg L-1 + NAA 5 mg L-1+ Vit B5, and Yellow Biloxi gave the highest number of proliferated somatic embryos compared to other genotypes. Increasing number of globular somatic embryo of all genotypes was obtained from the optimation of somatic embryo induction media being used, and Tanggamus genotype gave the highest number of globular somatic embryo which followed by Yellow Biloxi genotype. Tanggamus and Yellow Biloxi genotypes were also successfully formed the four steps of somatic embryos (globular, heart, torpedo, and cotyledonary stages), but in regeneration medium of MS0 and media MS + sukrosa 10 g L-1 + GA3 2 mg L-1 + BAP 4 mg L-1 + Vit B5 only Tanggamus genotype was regenerated into plantlet. <br /><br />Keywords: 2,4-D, NAA, somatic embryos, induction, proliferation<br /><br />