Mechanical stress-mediated acclimatization of in vitro regenerated plantlets is probably worth to be more investigated in future research since it serves as a non-chemical and cost-effective approach. Its expediency on potentially improving plants’ quality and reducing mortality percentage of plantlets provides an open field for future research.

1. Seminar
On
Plantlet Response to Seismomorphogenic Stimuli
(Mechanical stress-mediated acclimatization)
Presented By:
Dayashankar Singh
(M.Sc. Plant Biotech.)
17PBT004
Thigmo/seismomorphogenesis

2. “Acclimatization in life gives you true exhilaration…”
- Aswin

3. Outline
Introduction
What is seismomorphogenesisc stimuli (SMS)?
Why SMS is required in plant tissue culture?
Plantlet response to seismomorphogenesisc stimuli (SMS)
Limitation of seismomorphogenesisc stimuli (SMS)
Case study on Sansevieria trifasciata
Conclusion
Reference

4. Introduction
• In the last 10-15 years, micropropagation has shown spectacular development.
However, at present the widespread use of micropropagation is restricted for
several reasons, one of these being that a high mortality of plantlets in the
acclimatization stage.
• Due to the special environment in vitro, it is difficult to produce plants which
can be acclimatized to the outside environment.
• The term acclimatization is defined as the climatic adaptation of an organism,
especially a plant, that has been moved to a new environment (Conover and
Poole 1984).

5. Features of the tissue culture environment in the multiplication and rooting stages and their effects on
the growth of explants/plantlets in the multiplication, rooting, and acclimatization stages

6. Contd…
• Much research has been conducted to solve the problems related to acclimatization.
• Brainerd and Fuchigami (1981) examined responses of micropropagated plants to
low relative humidity.
• Wardel et al. (1983) examined the in vitro acclimatization of micropropagated plants
to humidity.
• Sutter and Hutzell (1984) used humidity tents and antitranspirants to increase the
survival percentage of micropropagated plants in the acclimatization stage.
• Read and Fellman (1985) developed a "controlled environment rooting facility" for
accelerating the acclimatization of micropropagated woody ornamentals.
• Sarmast MK, Salehi H, Khosh-Khui M. (2014) Seismomorphogenesis: A novel
approach to acclimatization of tissue culture regenerated plants.
• Sarmast MK (2016) In Vitro Regenerated Plants Response to Seismomorphogenic
Stimuli.

7. Contd…
• In nature, plants as sessile organisms must respond to stimuli throughout their
lifecycle. Mechanical conditioning is a physical stimulation or stress, deliberately
applied, to manage plant growth and quality (Latimer 1991).
• Plant growth responses to tactile or contact stimuli have been termed
thigmomorphogenesis (Jaffe 1973), while responses to shaking or vibrational
stimuli have been termed seismomorphogenesis (Jaffe 1973).
• Reported that in vitro acclimatization was more successful than ex vitro
acclimatization as it provides a sufficient period for gradual exposure of
plantlets to external environment (Joshi et al 2006).

8. What is seismomorphogenesic stimuli (SMS)?
• SMS is a mechanical stimuli (shaking
or vibration), in which plantlets are
indirectly influenced by shaking or
vibration under in vitro condition.
• Mechanical stimulation is, in
principle, plantlets adjust
physiologically and anatomically it
self by the process of
morphphysiological and molecular
change lead to acclimatization.
Stimuli

9. Why SMS is required in plant tissue culture?
• Prepare explants against stresses of the outside environment, because In vitro
growing plantlets usually contain malformed and unresponsive stomata and
poor epicuticular waxes.
Poor epicuticular waxes in in vitro
leaves
Abnormal, collapsed, completely closed stomata
with malformed guard cells in in vitro leaves
B

10. Contd…
• Poorly differentiated leaf structure,
• Poorly developed chloroplast,
• Supplied carbohydrate to independent
carbon fixation.
Chloroplast

11. Contd…
• A cost-effective and non-chemical approach could potentially improve plants
quality and reduce mortality percentage.

12. Plantlet response to SMS
 Morphophysiological Changes:
 PGRs
• Inter- and intracellular signaling components, including hormones and
potential second messengers, have been implicated in seismic induced
alterations in plant morphogenesis.
Stimuli
Ca2+ sensor
TCH3
Regulation of
PID
Regulate
auxin
regulator
Auxin
signaling and
morphogenic
response
Fig 1. Stimuli pathway of auxin

13. Contd…
 Proline
• The molecular mechanisms of how proline protects cells during stress are not
fully understood but appear to involve its chemical properties and effects on
redox systems such as the glutathione (GSH) pool.
• The higher proline levels reduced protein aggregation and
thermodenaturation.
• In an in vitro experiment, proline (1 M) protected nitrate reductase under
osmotic, metal, and H2O2 stress.

14. Fig 3. Potential functions of proline and proline metabolism in stress protection.

15. Contd…
 Nitric oxide
• Nitric oxide (NO) - a colorless gas - accumulation was observed in tobacco
suspension-cultured cells subjected to mechanical stress.
• Nitric oxide (NO) is involved in regulating various developmental and
physiological processes in plants, including seed germination, cell
differentiation, transition to flowering, and senescence .
• Since mechano-stress causes intracellular Ca2+ fluctuations as well as
transcriptional modification of genes in plant cells.

16. Fig. 4. Stress-induced signal transduction pathways via NO.
Mechano
-stress

17. Contd…
 Reactive oxygen species and Ca2+
• Cells may utilize ROS as signaling molecules to regulate the expression of
genes (Van Breusegem et al., 2001).
• ROS and Ca2+ are two key elements that rapidly induce upon mechanical
stress.
• Recently, Mori and Schroeder (2004) reported that ROS may control Ca2+-
permeable channel activity, thus suggesting a role for ROS in intracellular
Ca2+ regulation.

18. Fig. 5 A proposed model in which calcium and its sensors mediate mechano-
stimulations to mechano-responses

20. Contd…
 Molecular changes
• In response to thigmo/seismomorphogenesis, many genes with functions
such as calcium sensing, cell wall modifications, and also defense
antioxidant-related genes have been affected (Chehab EW, Wang Y, Braam J.
2011).
• Conserved touch–inducible plant genes are called TCH and have been
discovered by Braam and Davis (Braam J, Davis RW (1990).
• In addition, TCH4 encodes a Xyloglucan endotransglucosylase/hydrolases
(XTHs) which are cell wall modifying proteins involved in mechanical
stress.

21. Fig 6. Hypothetical model of thigmo/seismomorphogenesis and signal
transduction in plant cell
Stimuli

23. Limitation of SM
• The lack of specially designed equipment for shaking the in vitro plantlets in
large scale is a constraint.
• Plantlet must be served based on the species, its developmental stages and
sensitivity to stimuli.
• Plantlets are too sensitive to be able to endure thigmo/seismomorphogenetic
treatments, and therefore may be hurt.
• In vitro derived plantlets do not have complete control over their
evapotranspiration machinery system, thereby losing their water potential.

24. Case study on Sansevieria trifasciata

25. Abstract
Plantlets under in vitro conditions transferred to ex vivo conditions are exposed to biotic
and abiotic stresses. Furthermore, in vitro regenerated plants are typically frail and
sometimes difficult to handle subsequently increasing their risk to damage and disease; hence
acclimatization of these plantlets is the most important step in tissue culture techniques.
An experiment was conducted under in vitro conditions to study the effects of shaking
duration (twice daily at 6:00 a.m. and 9:00 p.m. for 2, 4, 8, and 16 min at 250 rpm for 14
days) on Sansevieria trifasciata L. as a model plant. In explants that received 16 min of
shaking treatment, leaf length and area and photosynthesis rate were increased
compared with control plantlets. Six months after starting the experiment, control
plantlets had 12.5 % mortality; however, no mortality was observed in other treated
explants. The results demonstrated that shaking improved the explants’ root length and
number and as a simple, cost-effective, and non-chemical novel approach may be
substituted for other prevalent acclimatization techniques used for tissue culture
regenerated plantlets. Further studies with sensitive plants are needed to establish this
hypothesis.

26. Materials and methods
 Mechanical stress treatment
• Uniform plantlets were selected and used for shaking treatments in the jar
glasses contain half-strength MS medium.
• Explants were shaken in a horizontal plane using a shaker (Peco. Pooya
Electronic, Iran). Explants in culture vessels, within a given experiment were treated
twice daily at 6:00 a.m. and 9:00 p.m. for durations of 0 (control), 2, 4, 8, and 16
min at 250 rpm for 14 days.

27. Contd…
• After applying the treatments, roots of explants were washed to remove agar and
then transferred to a soil mixture (perlite:peat:loamy soil, with the same volume)
and maintained in greenhouse under natural light (800 lmol m-2 s-1) at a day
temperature of 27 ± 5 C and RH of about 55 ± 5 %.

28. Contd…
 Measurement
• In explants that received 16 min of shaking treatment, leaf length and area and
photosynthesis rate were measure.
• Measurements were performed on clear sunny days between 10:00 a.m. to 1:00
p.m. (time of highest photosynthetic rate).
• Data were analyzed using one way ANOVA.

29. Fig. 7 Effects of mechanical shaking on photosynthetic (left) and proline content
(right) of Sansevieria trifasciata L., 6 months after shaking. Data represent the mean
± SD
Proline measured by using the
method of Bates et al. (1973).
Measured by portable photosynthesis
meter (Lci, ADC, UK).

30. Fig. 8 Effects of mechanical shaking on leaf area (left) and leaf length (right) of
Sansevieria trifasciata L., 6 months after shaking. Data represent the mean ± SD
Leaf area measured by a leaf
area meter (Delta-T. Devices Ltd)

31. Fig. 9 Thigmo/seismomorphogenesis of Sansevieria trifasciata L.
plants, 6 months after shaking at 250 rpm for 14 days

32. Fig. 10 Stomatal situation of Sansevieria trifasciata L. leaf after shaking at 250
rpm. a Tissue cultured explant stomata 4 min after shaking at 250 rpm. b Control
in vitro explants. c Acclimatized plant, 6 months after shaking.

33. Conclusion
Mechanical stress-mediated acclimatization of in vitro regenerated plantlets is
probably worth to be more investigated in future research since it serves as a
non-chemical and cost-effective approach. Its expediency on potentially
improving plants’ quality and reducing mortality percentage of plantlets
provides an open field for future research.

34. Reference
• Sarmast MK, Salehi H, Khosh-Khui M. (2014). Seismomorphogenesis: A novel approach
to acclimatization of tissue culture regenerated plants. 3 Biotech. 4: 599-604.
• Latimer JG, Thomas PA (1991) Application of brushing for growth control of tomato
transplants in a commercial setting. Horttechnology 1:109–110
• Appleton. Jaffe MJ (1973) Thigmomorphogenesis: the response of plant growth and
development to mechanical stimulation. Planta 114:143–157
• Joshi P, Joshi N, Purohit SD (2006) Stomatal characteristic during micro propagation of
Wrightia tomentosa. Biol Plant 50:275–278
• Z Li, Gong M. (2013). Mechanical stimulation-induced chilling tolerance in tobacco
suspension cultured cells and its relation to proline. Russian Journal of Plant
Physiology. 60:149-154.
• Chehab EW, Wang Y, Braam J. (2011). Mechanical force responses of plant cells and
plants, in: Mechanical integration of plant cells and plants. Springer. 173-194.