Adventitious Shoot Growth

Adventitious shoots can be reliably produced from nodal segment and stem tip explants from Stevia plants. With proper plant growth regulators, direct shoot formation is possible bypassing the callus stage. Two groups of plant growth regulators are important for plant tissue culture - Auxins and Cytokinins. A higher proportion of Cytokinin favours shoot formation whereas a higher proportion of Auxin favours rhizogenesis or root formation. The following tables elucidate the different combinations of plant growth regulators used by different workers for adventitious shoot formation from nodal segment or shoot tip explants.  

After aseptic inoculation of the explants in the medium with appropriate plant growth regulators, the culture vessels are incubated under a controlled contamination free environment under artificial illumination. The light intensity is a very important factor for explant differentiation. Generally light intensities between 1000 to 3000 lux is optimal for shoot growth for stevia. Low light intensity favours callus growth. For light to reach properly to the explants, culture vessels with transparent caps are more suitable. The culture vessels are also to be spaced adequately for proper distribution of light. Fluorescent lamps are most commonly using in tissue culture growth rooms. These lights have a wide range of wavelengths (350-750 nm) and appears to be are of low quality for promoting plant growth. With the advent of light-emitting diode (LED) technology, impressive progress has been made in environmental controls and morphogenetic responses. A wide array of LED lights with different spectra are currently available for application as light source in tissue culture. LED colors or combinations commonly used for in vitro culture are white, red, blue, and mixture rates of blue and red. It has been reported that red light is important for shoot and stem elongation, phytochrome responses and changes in plant anatomy. In contrast, blue light is important in chlorophyll biosynthesis, stomatal opening, chloroplast maturation, and photosynthesis.

The temperature regime maintained in the tissue culture growth rooms is 28°C to 30°C. The preferable relative humidity range is 50% to 90%. Generally a photoperiod of 14 - 16 hours (14 - 16 hours light and 10 - 8 hours dark cycle) works best for stevia. 

Under optimal culture conditions, the shoot formation initiation occurs within a week. The culture is incubated for 4 - 5 weeks for optimal number of shoot growth. Growth of up to 20 shoots from a single explant is not uncommon.

Light Absorption Spectra of Chlorophylls

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Plant Growth Promoter combinations used by different authors 

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The successive stages of in vitro propagation of Stevia rebaudiana. (a) Direct regeneration of shoot from explant, (b) Initiation of multiple shoot formation, (c) Development of more number of multiple shoot, (d) Root formation from regenerated shoot (e & f) Hardening and establishment regenerated plants.

From Anbazhagan et al (2010) Ref ID 3

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Comparison of Shoot development with different cytokinins 

From Javad et al (2019) Ref ID 4

(A) Axillary node explant of Stevia rebaudiana cultured on MS medium, (B) Multiple shoot formation,

From Attaya, AS (2017) An Efficient Protocol Of Stevia rebaudiana Regeneration For Large-Scale Production; Egypt. J. Agron. Vol. 39, No.1 pp.117- 125

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In the growth room, when the lights go off, temperatures cool slightly, and culture vessels and lids shrink; when lights are turned on again, culture vessels and lids expand. As a result of these changes, air enters the culture vessels. The culture room, benches or shelves where the cultures are kept must therefore be kept as clean as possible. To reduce the chance of contamination, access to the culture room should be limited to essential inspections.

 

The cultures are to be checked after 3-5 days for contamination. If present, bacterial contamination may be seen after a few days. Colonies of bacteria often form at the base of the stems inside the culture medium. They appear as opaque zones that develop rapidly into a mass of various colours, often orange-pink, green-black or black. Although fungal contamination may be seen after a few days, it usually is not seen for a week or so. It appears as furry, fuzzy colonies with no clearly defined edges growing on the surface of the culture medium. Contaminated cultures are to discarded immediately. 

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Initiation of multiple shoot formation of Stevia rebaudiana (B) Development of more number of multiple shoot after 4 weeks

From Abdul Razzak et all (2014) Ref ID 1

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In vitro multiplication of S. rebaudiana from nodal explants on MS medium supplemented with BAP + Kin (1.5+1.0). A. After 15 days, B. After 30 days, C. After 45 days of culture.

From Deshmukh and Ade (2012) Ref ID 4