PENGARUH 2,4- D TERHADAP PENINGKATAN KUALITAS DAN KUANTITAS KALUS YANG DIINDUKSI DENGAN KOMBINASI AUKSIN DAN SITOKININ PADA KULTUR DAUN RUMPUT BENGGALA ( Panicum maximum) VARIETAS TRICHOGLUME

To determine the effect various concentration 2, 4, 6 and 8 mg/l of 2,4- D (2,4- dicloropenoxy acetic acid) on callus induction of Benggala grass variety Trichoglume leaf culture on Murashige and Skoog (MS) standard medium and its organogenesis stimulated using deffent concentration of growth regulator, namely [N6- (2- isopenteny)- adenine] or 2iP (0, 0.2 and 1.0 mg/l ) and (l- naphthalene acetic ent, wacid) or NAA (0, 0.03, 0.16 and 3.0 mg/l) were performed. Percentage of callus were measured and organogenesis from callus were subjected to description analysis . The results showed that callus induction was optimum when 2,4-D was treated at 4 mg/l, friable callus were produced. Percentage production of callus week 4 was 46.2 percent, while using 8 mg/l 2,4 –D the callus production was about 59.7 percent yellowish coloured and more compact callus were produced. Combination of 0 mg/l 2iP ( auxin) + 3mg/l (cytokinin) at 3 weeks showed reseilted 100 % of calluses produced roots, the highest amaunt roots (9,0) was observed in combination 0 mg/l 2iP + 3 mg/l NAA, and the longest root (17,0 mm) was recorded in combination 0 mg/l 2iP + 3 mg/l NAA. Calluses yielded varied from white, yellowish to brown colour.

Sterile Tissue Preparation and Callus Induction of Curcuma longa Linn.

Curcuma longa Linn. (family Zingiberaceae), commonly known as ‘turmeric’, is native to Southeast Asia. Turmeric has been used for color, flavor as a spice in cuisine and employed for treatment of various diseases. The major component in yellow-pigmented fraction of turmeric is curcuminoids. Curcuminoid production in callus of C. longa Linn. is our focus of study. Sterile techniques to obtain germ-free of C. longa Linn. explants were investigated and the results showed that immersing rhizome buds in 70% ethanol for 5 min, followed by 0.10% HgCl2 for 10 min offered approximately 66% survival rate. Multiple shoots were generated from the aseptic rhizome explants cultured on Murashige and Skoog (MS) agar medium fortified with 3.00 µM of 6-Benzylaminopurine (BA) and 0.50 µM of 1-Naphthaleneacetic acid (NAA) at 25 ± 2°C under a photoperiod of 16 h light and 8 h dark. The sterile leaf sheath and root were subsequently used for callus induction which produced various responses when cultured on MS agar medium fortified with different concentrations of 2,4-dichlorophenoxy acetic acid (2, 4-D), Thidiazuron (TDZ) and BA. The highest induction yields of friable callus were obtained from leaf sheath segments cultured on MS agar medium fortified with 0.50 mg/l 2, 4-D which are the conditions proposed for successful production of callus culture of C. longa Linn. Keywords: Callus induction, Curcuma longa Linn., Turmeric, Plant tissue culture

Effects of Plant Growth Regulators and Sugars on Ehretia asperula Zoll. et Mor. Cell Cultures

Background: Belonging to the Boraginacae family, Ehretia asperula Zoll. et Mor., called “Xa den”, is a precious medicinal plant also known as the “cancer tree” by the Muong ethnic group in Hoa Binh Province, Vietnam. Xa den has been demonstrated to inhibit the development of malignant tumors, reduce oxidation and enhance the human immune system. This research focused on examining friable callus induction from young stems of Ehretia asperula Zoll. et Mor. Methods: Samples of Xa den were less than two years old. Young stems with 2 to 6 leaves served as explants for callus induction. Explants placed on autoclaved B5 nutrients incubated at 25oC, in the dark. The testing factors were concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) and Benzyl adenine (BA), types and concentrations of sugars.Result: Friable callus was induced on B5 medium with 0.4 mg/L of 2,4-D, 0.1 mg/L of BA and 30 g/L of glucose at the highest rate (100%). Additionally, callus grew best after 5 weeks of culture weighing 0.194 g. Friable callus was used as material for cell suspension cultures. After two weeks, the Xa den cell suspension cultures contained single cells and small cell clumps. The liquid medium had changed from dark yellow to light brown.

Explants selection for in vitro propagation of Pachyrhizus erosus L.

Pachyrhizus erosus tuber is rich in protein asides its agronomical value as a legume, but the seeds by which it is propagated have very low viability. This study established sterilization protocol and effect of various concentrations of auxins and cytokinins on callus production and shoot regeneration from explants of P. erosus. Explants and seeds were sterilized using sodiumhypochlorite (NaClO) solution (5, 10 and 15% v/v) for 5 and 10 mins. Nodal, stem and leaf explants from in vitro germinated P. erosus and tuber from field grown plant were sterilized and cultured on Murashige and Skoog (MS) medium (control) and MS combined with different concentrations of auxins (NAA and 2, 4-D) and cytokinin (BA and Kinetin) and the cultured explants were monitored in terms of degree of callus formation, morphology and colour of callus and also for shoot induction. The results showed that seeds of P. erosus sterilized with 10% NaClO solution for 10 mins and germinated in vitro is the best way of getting sterile nodal, stem and leaf explants for the in vitro propagation of the plant, while tuber explants could be sterilized with 15% NaClO for 10 minutes. Nodal explants inoculated in MS medium supplemented with 1.0 mg/L BA gave the highest shoot regeneration response, while stem explants inoculated on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA also gave the highest amount of friable callus. The study concluded that in vitro germinated seeds were the best way of getting explant for P. erosus.

Effects of different factors on friable callus induction and establishment of cell suspension culture of Hovenia dulcis (Rhamnaceae)

Medicinal plants are an important therapeutic option for a large share of the world’s population. To establish an in vitro culture system for the production of secondary metabolites from Hovenia dulcis, we studied the effect of auxins, cytokinins, absence of light, and silver nitrate on the development of friable callus. Callus cultures were established for the first time and used to obtain cell suspension cultures. Supplementation with KIN (Kinetin) produced calli with both compact and friable areas, while the addition of TDZ (Thidiazuron) only produced compact callus. The maintenance of cultures in the dark induced a slight enhancement on friability when the auxin PIC (Picloram) was present in the culture medium. The addition of silver nitrate promoted the formation of friable calli. Dry weight analysis showed no significant differences in biomass growth, and, therefore, 2.0 mg.L-1 was considered the most suitable treatment. The presence of silver nitrate was not required for the establishment of cell suspension cultures. Dry weight analysis of cell suspensions showed higher biomass production in the absence of silver nitrate. PIC promoted 100% of cell suspension culture formation in the absence of silver nitrate, and higher biomass production was observed with the lowest concentration (0.625 mg.L-1). No morphological differences were observed among the different concentrations of PIC. Phytochemical screening showed the presence of saponins, flavonoids, flavonols and catechins in the extracts obtained from H. dulcis calli. These results show that the cell cultures herein established are potential sources for the production of H. dulcis secondary metabolites of medicinal interest.

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.

Establishment of a Cell Suspension Culture of Eysenhardtia platycarpa: Phytochemical Screening of extracts and Evaluation of Antifungal Activity.

Eysenhardtia platycarpa (Fabaceae) is a medicinal plant used in México and it lacks biotechnological studies for its use. The aim of this work was to establish a cell suspension cultures (CSC) of E. platycarpa, determine the phytochemical profile, and evaluate its antifungal activity. Friable callus and CSC were established with 2 mg/L 1-naphthaleneacetic acid plus 0.1 mg/L kinetin. The highest total phenolics of CSC was 15.6 mg GAE/g dry weight and the total flavonoids content ranged from 56.2 to 104.1 µg QE/g dry weight. CG‒MS analysis showed that the dichloromethane extracts of CSC, sapwood and heartwood have a high amount of hexadecanoic acid (22.3 ‒ 35.3 %) and steroids (13.5 ‒ 14.7%). Heartwood and sapwood defatted hexane extracts have the highest amount of stigmasterol (≈ 23.4%) and β-sitosterol (≈ 43%), and leaf extracts presented β-amyrin (16.3%). Methanolic leaves extracts showed mostly sugars and some polyols, mainly D-pinitol (74.3%). Dichloromethane and fatty hexane extracts of CSC exhibited the percentages inhibition higher for Sclerotium cepivorum with 71.5 and 62.0%, respectively. The maximum inhibition for Rhizoctonia solani was with fatty hexane extracts of the sapwood (51.4%). Our study suggest that CSC extracts could be used as a possible complementary alternative to synthetic fungicides.

Effects of plant growth regulators, mineral medium, and pH values on the callus induction of young stem cutting slices of Ehretia asperula Zoll. et Mor.

Xa den (Ehretia asperula Zoll. et Mor.) belongs to the Boraginaceae family, which is a precious medicine, called “cancer” tree in Muong at Hoa Binh province traditional medicine. Xa den was shown to inhibit the development of malignant tumors, reduce oxidation, enhance resistance... In this study, we investigated the induction of friable callus in Xa den young stem section that could be further used to the culture cell suspension and produce bioactive compounds. In that, we focussed on the effects of the plant growth regulators, mineral media, and pH on the formation of friable callus of Xa den. The results showed that all treatments stimulated the formation of callus from Xa den thin stem (0.5-1.0 mm). In particular, samples which were cultured in B5 medium supplemented with 0.4 mg/L 2,4-D and 0.1 mg/L BA, at pH 5.8 produced the highest percentage of secondary callus in friable (85%), and this callus also had highest fresh weight after 4 weeks of culture (0.054 g). To evaluate the presence of natural compounds in the callus and compared to those in cultivated plants, the ethanol extract of dry leaves, branches, and fresh callus were used. The results showed the presence of natural compounds in the callus such as organic acid, phenolic, tannin, alkaloid, and flavonoid was similar to the one in a 2-year-old plant.