Artificial Seed Production from Encapsulated Microshoots of Cauliflower (Brassica oleraceae var botrytis)

A cost effective protocol for the production of cauliflower microshoots suitable for encapsulation was designed. Microshoots were encapsulated in sodium chloride matrices. The use of 2% of sodium alginate and 15 g/L of dehydrate calcium chloride produced the optimal quality of artificial seeds (rigidity, conversion rate and viability). Of the various plant growth regulator combinations used with the microshoot liquid culture medium, the use of 1 mg/L of IBA (indole butyric acid) and 1 mg/L Kinetin was found to be optimal in terms of the conversion rate and viability of artificial seeds. To standardize a medium composition of artificial endosperm of synthetic seeds, different concentrations and combinations of plant growth regulators with S23 (4.4 MS + 30 g/L sucrose) medium were used in the beads to achieve optimum conversion rate and viability on an in-vitro medium. Whilst several combinations of plant growth regulators gave a conversion rate up to 100% (for example (0.5 mg/L Kinetin + 0.5 mg/L IBA), (1 mg/L Kinetin + 0.5 mg/L NAA (naphthaleneacetic acid)) and (1 mg/L Kinetin + 1 mg/L IAA (indole-3-acetic acid)), no significant effect on the viability of artificial seeds was found when these combinations were used. Artificial seeds were cultivated in a semi-solid medium containing several types and concentrations of auxin, 2 mg/L of IBA gave the best results in terms of artificial seed viability. However, artificial seed conversion rate was not significantly affected by the auxins and full conversion rate was obtained using many different treatments. This research indicated the feasibility of using artificial seeds as a promising alternative to seeds produced by traditional methodology

Water and Chlorophyll Content and Leaf Anatomy of Patchouli Planlet (Pogostemon Cablin Benth.) Resulted by Shoot-tip Culture Experience Hyperhydricity After Treatment of Modification Ammonium Nitrate or Macro Salt Concentration on MS Medium (Murashige Skoog)

Hyperhydricity is a symptom of abnormal morphological and physiological function which inhibits the regeneration of plantlets. In general, the main symptom of hyperhydricity is a change in the condition of the plantlets which looks clear (Glassy) as a result of low levels of chlorophyll, the high water content in the plantlets, and the abnormal anatomical structure of the leaves. Hyperhydricity can be controlled by reducing cytokinin concentration, increasing gelling agent concentration, and reducing ammonium nitrate and macro salt concentration on medium. Objective of this research was to reduce hyperhydricity in shoot tip culture of patchouli by modification of ammonium nitrate and macro salt concentration on MS medium. The various treatment concentrations of ammonium nitrate were 0 mg.L-1 (0), 41.25 mg.L-1 (¼ concentration), 825 mg.L-1 (½ concentration), 1650 mg.L-1 (1 concentration) and macro salt MS with 0, ¼ MS, ½ MS, MS with 5 replications. Hyperhydricity on patchouli shoots could be lowered, as indicated by the decrease in water content from 96% to 90-91%, the increase in total chlorophyll content, and the increased number of palisade cells and stomata on the leaf treatment outcome. The concentration treatment of ammonium nitrate showed better results than the concentration of macros salt in increasing the total chlorophyll content, but it did not differ significantly in lowering water levels and increasing the number of palisade cells and stomata. ¼x concentration treatment of ammonium nitrate could increase chlorophyll content of 0.16 to 0.97 mg.g-1, but MS with 1x concentration showed the best result in the increase of number of palisade cells and stomata of the leaves

Establishment of in vitro tissue cultures from Echinacea angustifolia D.C. adult plants for the production of phytochemical compounds

SUMMARY The establishment of in vitro cultures of Echinacea angustifolia D.C. was obtained directly from sections of flower stalks of adult plants. The shoot formation was obtained from this plantmaterial placed on a modified MS basal medium named CH supplemented with 0.5 mg L1 6 benzylaminopurine (BA). The in vitro propagation procedure of E. angustifolia consisted of three distinct phases: an initial regeneration phase fromstalk sections (IP shoots on basal mediumwith 0.25 mg L1 BA), an elongation phase on active charcoal and an axillary proliferation of the shoots (AP shoots on basal medium with 0.5 mg L1 BA).Regenerating calli were established from leaves of in vitro shoots cultured on CH medium supplemented with 3 mg L1 BA and 0.5 mg L1 indole-3-butyric acid (IBA). Developed shoots from the callus cultures were subcultured on the CH medium with 0.5 mg L1 BA (leaf regenerated shoots: LR shoots). The secondary metabolite content of the in vitro plant material was compared with that of the greenhouse growing plants. The quali-quantitative LC-DAD-ESI-MS analysis on the extracts from axillary proliferation shoots (AP shoots) showed significant production of caffeic acid derivatives while leaf callus and LR shoots, accumulated mainly alkamides. These results showed that the proper choice of the procedures for in vitro multiplication allowed us to obtain plant biomass able to produce the active compounds typical of E. angustifolia plants

Effect of Permeable Vessel Closure and Gelling Agent on Reduction of Hyperhydricity in in Vitro Culture of Carnation

Hyperhydricity, an abnormal morphological appearance and physiologicalfunction, is an important problem in carnation tissue culture. The problem causes premature flowering, high occurrence of abnormal shoots, difficulty in transferring hyperhydric plantlets to soil, and low survival rate of plantlets. High relative humidity and the water potential are considered as the key factors involved in the abnormality. Furthermore, permeable culture vessel and gelling agent were assured to be high potential treatment to eliminate it. Objective of this research was to reduce hyperhydricity in regenerants of carnation using different permeable vessel closures and gelling agents and to assess the multiplication and acclimatization abilities of recovered shoots. Experiment was arranged in randomized complete block design with four replications. First factor was different types of closure, i.e. cotton wool, plastic wrap, parafilm and aluminium foil, while second one was gelling agents, i.e. bacto agar, phytagel, swallow agar, and Type 900 agar. The recovered shoots were then multiplied, rooted, and acclimatized. The results showed that hyperhydricity was successfully reduced by applying permeable closure (cotton wool and plastic wrap) in combination with Type 900 agar. The combination of plastic wrap and Type 900 agar was the most appropriate treatment in reducing hyperhydricity and producing good quality shoots. The treatment reduced the problem down to 23% of total condition of hyperhydricity (100%) and increased leaf chlorophyll content from 0.0883 to 0.1288 mg mg-1. The plastic wrap was easily applied and cheaper material compared to cotton wool. The recovered shoots were able to produce 1-3 healthy axillary shoots and easily rooted on half-strength MS. The recovered plantlets were simply acclimatized with survival rate up to 100% on kossas peat + soil (1:1, v/v) and flowered 4-5 months after acclimatization with decreasing in number and size of flower

In vitro shoot regeneration from preconditioned explants of chickpea (Cicer arietinum L.)

The present study reports the successful shoot regeneration of preconditioned mature embryo and embryonic axis explants of chickpea cv. Gokce. Explants were preconditioned with 10 mgl benzylaminopurine (BA) for 7 days followed by culture on Murashige and Skoog (MS) medium containing 0.25, 0.50, 1.00 and 2.00 mg/l BA with or without 0.25 mg/l naphthalene acetic acid (NAA) supplemented with 4 mg/l activated charcoal and 1 mg/l polyvinylpyrrolidon (PVP). Shoot regeneration was recorded on all explants. Maximum number of (14.75) shoots per explants on mature embryo were recorded on MS medium containing 2.0 mg/l BA with 0.25 mg/l NAA. Whereas, 16.83 shoots per explants were recorded on MS medium containing 2.0 mg/l BA. Presence of NAA in the culture medium decreased the mean shoot length of both explants compared to medium devoid of 0.25 mg/l NAA. Regenerated shoots were rooted on MS medium containing 1.0 mg/l indole-butyric acid (IBA) after 4 weeks of culture. Rooted plantlets were transferred to pots for acclimatization under green house conditions

Development of Micropropagation System and Reduction of Hyperhydricity in Regenerants of Carnation (Dianthus Caryophyllus L. Cv. Maldives)

This study was carried out with the main objectives of developing a micropropagation system for Dianthus caryophyllus cv. Maldives and reducing hyperhydricity for healthy shoot production. The development of a micropropagation system included selection of explant and combination-concentration of growth regulators, optimization, multiplication of shoots, rooting and acclimatization. Hyperhydricity study included selection of types of closure and gelling agents, application of ventilated culture vessel, multiplication of recovered shoots and acclimatization of recovered plantlets. The experiment was factorial arranged in a randomized complete block design with four replications. Each treatment consisted of twelve explants per replicate. In axillary proliferation of shoots using two types of explant and five combinationconcentrations of growth regulators, node explant placed on MS medium containing 1.0 mg/L BA and 0.1 mg/L NAA was the most suitable combination in stimulating high axillary shoot production with low rate of hyperhydricity. Lowering the concentration of NAA from 0.1 mg/L to 0.05 mg/L in combination with 1.0 mg/L BA in the optimization experiment improved axillary shoot production from 4.9 to 5.6 shoots per explant and reduced hyperhydricity to less than 30%. In adventitious shoot formation from three explants placed on five concentrations of BA and NAA, the first young and fully developed leaves placed on MS medium supplemented with 0.1 mg/L BA and 0.01 mg/L NAA was the most suitable combination in inducing high adventitious shoot formation (43.3%) with lower hyperhydricity (60.0%) compared to other combinations tested. MS medium containing 1.0 mg/L BA with 0.05 mg/L NAA and 0.5 mg/L BA with 0.1 mg/L NAA were the most appropriate media in inducing high shoot multiplication, whereas MS medium supplemented with 0.1 mg/L BA with 0.02 mg/L NAA and 0.1 mg/L BA with 0.01 mg/L NAA were most suitable in producing good quality shoots for rooting. High production of good quality shoots were produced only after the first subculture and reduced in the subsequent subcultures

The role of silicon in plant tissue culture

Growth and morphogenesis of in vitro cultures of plant cells, tissues and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si) is the most abundant mineral element in the soil. The application of Si has been demonstrated to be beneficial for growth, development and yield of various plants and to alleviate various stresses including nutrient imbalance. Addition of Si to the tissue culture medium improves organogenesis, embryogenesis, growth traits, morphological, anatomical and physiological characteristics of leaves, enhances tolerance to low temperature and salinity, protects cells and against metal toxicity, prevents oxidative phenolic browning and reduces the incidence of hyperhydricity in various plants. Therefore, Si possesses considerable potential for application in a wide range of plant tissue culture studies such as cryopreservation, organogenesis, micropropagation, somatic embryogenesis and secondary metabolites production