Suitability of different fruit crops under different stress conditions

1. Suitability of different fruit crops under
different stress conditions
Presented by:
Mandeep Kaur
PhD Fruit Science
Presented to:
Dr H S Dhaliwal
Professor of Horticulture

2. What is Stress ?
• Stress in biology is any change in environmental conditions that might
reduce or adversely change plant growth and development (Levitt, 1972).
• Stress is a condition caused by the factors that tends to alter an
‘equilibrium’ (Strasser, 1998).
• Jacob Levitt- Father of plant stress physiology.
• One stress is often combined with typical set of other stresses, so they do
not act individually in nature.
• Stresses may be additive or mitigating each other.

3. Types
of
Stress
Biotic
Abiotic
Temperature
High
Low
Water
Excess
Deficit
Salt/Ion
Toxicity
Deficiency
Strasser (1998)

4. Physiological adaptations under different stress conditions
• Plants can modify their life cycles: Eg temperate fruits
• Phenotypic changes in leaf structure and behaviour are important stress
responses (changes in leaf area, leaf orientation, trichomes, and the cuticle).
• Under conditions of water, heat, or salinity extremes, leaves may be narrower or
may develop deeper lobes during development.
• Plants change stomatal activity in response to dehydration stress.
• Plants adjust osmotically to drying soil by accumulating solutes.
• Salt exclusion: Prevents the entry of salts into the vascular system. Eg Vitis
champini (Cl-ion excluder) and Cleopatra mandarin
• Salt elimination: Salt-secreting glands and hairs actively eliminate salts, thus
keeping the salt concentration in the leaves beneath a certain threshold.
• Heat shock proteins can be induced by different environmental conditions: In
response to sudden 5 to 10°C increases in temperature, plants produce a unique set
of proteins referred to as heat shock proteins (HSPs).
• Many plants have the capacity to acclimate to cold temperature: Several
specialized plant proteins, termed antifreeze proteins, limit the growth of ice
crystals through a mechanism independent of lowering of the freezing point of
water.

5. Plant Response to Different Stress
• Phytohormones like ABA, SA, JA, and ET accumulates and increase in plant
tolerance to various stress.
• ABA is responsible for plant defense against abiotic stresses such as drought,
salinity, cold, heat stress and wounding.
• SA, JA and ET play major roles in response to biotic stress conditions as their
levels increase with pathogen infection.
• Role of different proteins in stress response: Heat Shock Proteins, Anti free
proteins, DREB proteins, LEA proteins and PR proteins.
• Osmoprotectants: Osmotin accumulates in salt adapted cells, the
overexpression of osmotin induces proline accumulation (a compatible
solute).

7. A scheme representation of abiotic stress at the cellular level

8. Biotic stress at the cellular level

9. A scheme for the interaction interface and overlapping signaling pathways of
abiotic and biotic stress at the cellular level

10. A scheme for the effects of abiotic and biotic stress at the plant level

11. Approaches for building combined abiotic and biotic stress Tolerance in plants

12. Beyond Chemical Triggers: Evidence
for Sound-Evoked Physiological Reactions in Plants
• Naturally occurring and artificially generated sound waves contribute to
plant robustness.
• New information is emerging about the responses of plants to sound
and the associated downstream signaling pathways.
• This paper provide an overview of the latest findings, limitations, and
potential applications of sound wave treatment.
• No doubt, chemical triggers improves plant health by enhancing plant
growth and resistance.
• But, Sound wave treatment is a new trigger to help protect plants
against unfavorable conditions and to maintain plant fitness by
modulating physiological traits. It shows adaptive advantage in plants.
Jung et al (2018)
Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea,

13. Sound production and perception in plants
(A) Sound production: Plants produce sound vibrations in their xylem via the
generation of tension in the xylem vessel when its diameter decreases.
Additionally, gas bubbles produced in xylem vessels during transpiration may
produce sound.
(B) Sound Perception: Although there are no visible alterations, transcriptional
and translational changes occur in plants exposed to sound vibrations. But, levels
of stimulus responsive, signaling-related, redox homeostasis, and defense related
transcripts are changed in sound-exposed plants.
However, the specific organs or proteins used for sound perception have not yet
been identified.
PRODUCTION (SPEAKING)
AND
PERCEPTION (LISTENING)
OF SOUND IN PLANTS
Can Plants Make
Sound Vibration?
Can Plants Respond to
Sound?

14. Responses of plants to sounds of different frequencies and magnitudes.

15. 1. Sound promotes growth by increasing IAA and GA.
2. Sound pretreatment enhances plant immunity against subsequent pathogen attacks by activating
the plant defense hormones SA and JA.
3. Sound treatment triggers drought tolerance by changing the elasticity and flexibility of the
cell wall, which affects the ability of plants to absorb water.
4. Ethylene production is delayed by down-regulation of E biosynthesis and expression of
signaling-related genes.
5. Sound treatment increases expression of photosynthesis-related genes, such as those encoding
fructose 1,6-bisphosphate aldolase and the rubisco small sub-unit, and may induce CO2 fixation
Sound waves as a
plant stimulant and
protectant.
Artificial sound
treatment can elicit
various effects in
plants.

16. Perspectives And Remaining Questions
Sound represents a potential new trigger for plant protection
1. The discovery of an organ or a specific protein in plants that recognizes sound
waves would help us maximize the effectiveness of the use of sound treatment
in field trials.
2. Second, technology used to engineer sound quality, such as the fine turning,
modification, and mixing of sounds, must also be improved to facilitate its use for
sound-mediated stress relief and increased plant growth.
3. Third, the analysis of plant biomarkers such as Pathogenesis-Related 1 protein
(PR1) (for systemic acquired resistance) will help scientists optimize sounds to
maximize sound-specific plant stress relief.
4. Side effects of sound waves. Humans can differentiate and recognize sounds
ranging from 20 to 20,000 Hz. If the sound vibration used to treat plants causes
damage to animals, humans, or microbes after long-term exposure, a detailed
examination and evaluation of the effects of various exposure times and high-
frequency (e.g., above 20,000 Hz) will be required.
• If the proper electric power supply, speakers, and associated sound-generating
equipment are utilized sound treatment can constitutively be applied for long
periods of time without additional input.
• This unique setup, which has not been tested before, awaits your next
experiment.

17. SUITABILITY OF DIFFERENT FRUIT CROPS
UNDER
SALT STRESS CONDITIONS

18. Salinity Tolerance of Some Grapevine Cultivars as
Affected by Salt Creek and Freedom Rootstocks
• Objective: To study variability in salt tolerance of varieties (Flame seedless, Early
sweet, Superior seedless and Red Glob) grafted on two rootstocks (Salt creek,
Freedom) compared with own rooted vines on growth and nutrient content under
varying levels of salinity treatments (1000, 2000, 3000 ppm).
• Results: Vine growth significantly decreased with the increase in salinity levels in
all vines (Reason: Increasing salt concentration significantly reduced the N, P and K
contents in the leaf petioles).
• At the highest salinity level (3000 ppm), the survival vine percentage was zero %
for own rooted vines, except, the own rooted Flame seedless that recorded 50
percent of vine survival.
• Among the scion-rootstock vines, about 70-80% vine survival was observed with
the vines grafted on Salt creek and 60-65% in vines grafted on Freedom rootstock.
• Conclusion: All cultivars grafted on Salt creek rootstock were more tolerant to
salinity.
National Research Center, Egypt Desouky et al (2015)

19. Effect of saline irrigation water on vines height (cm)

20. Effect of saline irrigation water on survival vines percentage

21. Relative Salt Tolerance of Different Grape
Rootstocks to Different Chloride Salts
• Objective: To study the variability in salt tolerance of grape rootstocks (viz.,
Dogridge, Salt Creek, RS-19, SO4 and 1613-C) to different chloride salts viz., NaCl,
KCl, MgCl2 and CaCl2 salts) applied at 4 meq, 8, 16 and 32 meq/lt concentrations.
The mean of different concentration of salts was used for salt tolerance assessment.
• Observations: The relative salt tolerance was assessed based on growth
parameters, dry weight of root to shoot ratio and also on the ability of rootstocks to
limit uptake of Na and Cl ions.
• Results: Highest root growth parameters measured in terms of root number
(59.79), length (42.9cm) and dry weight (24.51g) and also dry weight of root to
shoot ratio (0.6) was recorded in Dogridge.
• High chloride exclusion ability represents the salt tolerance. Significantly
lowest chloride content was recorded with Dogridge (0.87%) rootstock whereas
the highest chloride content was recorded with Salt Creek (1.14%).
• The Cl accumulation in leaf was significantly higher when applied in the form of
NaCl followed by KCl.
• Conclusion: Due to low Cl‫־‬ content in leaves, high dry weight of root shoot ratio
and high vigour, Dogridge is more tolerant to salinity than other rootstocks studied.
Saritha et al (2017)Grape Research Station, Rajendranagar, Hyderabad

22. Effect of varying chloride salts on root parameters in different grape rootstocks

23. Effect of different chloride salts on Cl content (%) in different grape rootstocks
Chloridecontent(%)

24. Adaptation of grapevine (wild and rootstocks) to salinity
• Objective: To establish a possible correlation between
salt resistance and mineral content of plants after salt
treatments applied in two different culture systems, in
vitro culture of meristematic structures and potted
plants, respectively.
• EXP. 1: The shoot tips were harvested from potted
plants and the apexes were cultured on solidified
MURASHIGE and SKOOG (1962) medium, supplemented
with 17 (V1), 34 (V2) and 43 (V3) mM NaCl. The
medium without NaCl content was used as a control for
the test.
• EXP. 2: Potted plants were watered every two days with
solution of 51, 68, 102 mM NaCl, and also without NaCl
(as control) for 30 d.
Five rootstocks
SO4
Kober 5BB
140Ru
Fercal
Riparia Gloire
Six different wild plants from
Vitis vinifera L. ssp. sylvestris
Vs 1
Vs 10
Vs 9 DM
Vs 3 EB
Letea 1
Letea 2Popescu et al (2015)Romania

25. The influence of NaCl on explants evolution and shoots development, after
5 passages on the same media composition.
Example for rootstock 'Fercal 242' and wild plant Letea 1.
From the left to the right: control (M), 17 (V1), 34 (V2) and 43 (V3) mM NaCl.

26. The effects of different salt concentrations on plant development; potted
plants after 15 treatments.
From the left to the right: control (M), 51 mM (V1), 68 mM (V2) and 102 Mm
(V3) NaCl.

27. Effect of saline treatments on nutrients tissue content

28. In the potted conditions, the tested rootstocks reacted as “Na includes” while in vitro tests the
plants belonging to wild populations reacted as “Na excluders".
In vitro conditions favored absorption and retention of K and Na elements in vegetative
structures, while potted treatments increased the absorption of P.
Different NaCl resistance mechanisms are involved in vitro plant material and potted
plants subjected to salt stress.
On the whole, the wild grapevine individuals, in comparison to the rootstocks, expressed a
higher ability to adapt to the salt stress in both type of treatments.
Effect of saline treatments on nutrients tissue content

29. Grape Rootstock Response to Salinity, Water and
Combined Salinity and Water Stresses
A field experiment was conducted at the USDA ‐ California from June 2013 to Sept
2017 to evaluate the effect of 4 irrigation water salinity levels and three water
treatments on three grape rootstocks purported to be salt tolerant.
Objectives:
1. The salt tolerance (yield) response of Cabernet Sauvignon grafted on to three
rootstocks.
2. Response to irrigation quantities, from meeting crop evapotranspiration (ETc)
to application of 80% and 60% of crop evapotranspiration (ETc).
3. Vine and fruit yield response to combined effects of salt stress and irrigation
quantity.
The crop evapotranspiration (ETc) was measured by lysimeters
USDA‐Agricultural Research Service, U.S. Salinity Laboratory in Riverside, California
Suarez et al (2019)

30. 4 irrigation water
salinity levels
S 0 = 0.7 dS m−1 water
S 1 = 1.7 dS m−1 water
S 2 = 2.7 dS m−1 water
S 3 = 3.7 dS m−1 water
Cabernet Sauvignon scions
grafted onto
SC represents Salt Creek rootstock
Ru represents 140 Ruggeri rootstock
SG represents St. George rootstock
Three water treatments
D 0 = 100%
ETc
Two 1.0 L h−1 and one 0.5 L
drippers
D 1 = 80%
Etc
Two 1.0 L h−1 drippers
D 2 = 60%
ETc
One 1.0 L h−1 and one 0.5 L
h−1 drippers,

34. Conclusion
• The 140 Ruggeri scion was the top producer across all treatments
including control, followed by Salt Creek, with St. George significantly less
productive than 14 Ruggeri across all treatments.
• No significant rootstock differences related to water stress was found.
• Reductions in yield was observed under reduced water applications.
• Under saline conditions, the reduced water applications result in reduced
leaching and thus higher soil salinity.
• Thus, it is recommend that water application should not be reduced
under salinity conditions.

35. Salinity Soil Effects on Yield, Fruit Quality and Mineral Composition
of Superior Seedless Grapevines Grafted on Some Rootstocks
• Objective: To evaluated superior seedless grape cultivar grafted on Paullson 1103,
Salt Creek and freedom grapevine rootstocks to soil salinity compared to own rooted
superior seedless cultivar.
• Methodology: Vegetative growth parameters, chemical analysis, yield and berry
characteristics and antioxidant isozymes electrophoresis was estimated.
• Results: Superior grape cultivar grafted on three rootstocks gave the best results as
compared to own rooted superior grape cultivar for three consecutive seasons.
• Superior seedless grape cultivar grafted on Salt Creek rootstock had the highest
percentage of bud burst and fruitful buds, improved the best vegetative growth
parameters, increment depth of the roots and their distribution in the soil profile.
• Cane content of total carbohydrates, leaf content of total chlorophyll and mineral
content were increased and leaf prolin content, sodium and chloride were reduced.
Additionally, it had a positive impact on the yield and berry quality attributes.
• Conclusion: Superior seedless grafted on Salt Creek, Paullson 1103 and freedom
grape root stocks were more tolerant to soil salinity than superior seedless cultivar
on own rooted root stock, vines grafted on Salt Creek was the most tolerant one.
Agriculture Research Center, Giza, Egypt Ahmad (2016)

36. Effect of grafting superior seedless cv. on some rootstocks under saline condition on
minerals content during (2012, 2013 and 2014) seasons

37. Responses of Cherry Plant Grafted onto CAB-6P,
MaxMa 14 and Mazzard Rootstocks to Short Term Salinity
• Objective: To investigate morphological and physiological responses of a cherry
cultivar (0900 Ziraat) grafted onto three rootstocks (CAB-6P, MaxMa 14 and
Mazzard) to 35 mM NaCl stress.
• After one month, salt stress decreased plant growth of cherry plants. Rootstock,
scion and shoot diameters and shoot length significantly decreased as a result of
restriction in nutrient uptake due to the salinity that limits photosynthesis
consequently decreases plant tissue extension.
• Results: In the current study, the growth of Mazzard rootstock was less damaged
& showed higher tolerance to short term salt stress as compared to CAB- 6P and
MaxMa 14.
• Reason: Lower depression in plant growth, less decrease in chlorophyll content
and more stability in cell membranes as compared to other rootstocks.
• Conclusion: In the short term, cherry can be considered salt tolerant.
• But, long term responses of cherry trees to salt stress may be different due to the
salt accumulation in leaves.
Aras & Esitken (2019)Selcuk University Konya, Turkey

38. Effects of salinity on physiological responses of cherry plants
* Relative chlorophyll value was measured with a Minolta SPAD-502 chlorophyll meter.
* Stomatal conductivity and leaf temperature measurements by leaf porometer.
* Membrane permeability by the procedure of electrolyte leakage based on Lutts et al (1996).
* Leaf relative water content (LRWC) by the procedure of Smart and Bingham (1974).

39. Improved salt-tolerance in Citrus macrophylla
mutant rootstocks
• Mutagenesis by gamma rays was used to develop salt tolerant mutants.
• In previous works, five mutants (MM3B, MM4B, MM5B, MM2A and MM3A)
from Citrus macrophylla, obtained through the exposure of seeds to gamma
radiation (129 Gy), were selected for their ability to cope with salt stress in
vitro.
• In the present experiment, the selected mutants, along with Citrus
macrophylla shoots as control, were submitted to 60mM NaCl (MM3B, MM4B
and MM5B mutants) or to 80mM NaCl (MM2A and MM3A), in their respective
culture media.
• After 8 weeks of exposure, proliferation rates, shoot length and damaged
leaves were recorded to assess the effects produced by salinity in the culture
medium.
Jiménez and Tornero (2019)Murcia, Spain

40. Control in vitro MM2A mutant shoot (A)
MM2A mutant shoot exposed to 80mM NaCl (B)
‘Alemow’ mutant shoot exposed to 60mM of NaCl (C)
MM5B mutant shoot exposed to 60mM NaCl (D)
MM3B mutant shoot exposed to 60mM NaCl (E)
MM3A mutant shoot exposed to 80mM NaCl (F)
MM4B mutant shoots exposed to 60mM NaCl (G)

41. Effect of NaCl exposure on the NO ³‫־‬ content in ‘Alemow’ and the mutants MM3B,
MM4B, MM5B, MM2A and MM3A.
NO ³‫־‬ increased in the mutants, as compare to ‘Alemow’.
This could be the key to resisting stress, since sustaining the supply with essential nutrients,
such as nitrate, is believed to enhance plant salinity tolerance.
This is because N is involved in the biosynthesis of many important compounds and its lack
would cause a serious imbalance, resulting in reduced growth and lower yield.

42. Effect of NaCl exposure on the chlorophyll content in ‘Alemow’ and the mutants
MM3B, MM4B, MM5B, MM2A and MM3A.
Before salt stress (60mM), higher photosynthetic capacity was present, to produce and
store higher amounts of photosyntetates for use in stress conditions.
This capacity, along with the other genetic adjustments involved in mutation, could have
allowed these mutants to improve proliferation and growth despite the leaf damage, as
indicated by the productivity records in salinity.

43. Effect of NaCl exposure on the proline content in ‘Alemow’ and the mutants MM3B,
MM4B, MM5B, MM2A and MM3A.
Proline has been reported to act as a protector that is able to overcome abiotic stresses
through the balance of the osmotic potential.
But, here, the relation between its accumulation and stress is controversial and remains
unclear.

44. Effect of NaCl exposure on the soluble sugars in ‘Alemow’ and the mutants
MM3B, MM4B, MM5B, MM2A and MM3A.
Soluble sugars & starch maintains the osmotic adjustment in the plant.
Here, soluble sugars content augmented in MM5B explants from control to salinity, which shows
better response to the osmotic stress in this mutant.

45. Effect of NaCl exposure on the starch in ‘Alemow’ and the mutants MM3B,
MM4B, MM5B, MM2A and MM3A.
• Starch increases in salinity conditions in order to regulate osmotical disequilibrium in the
plant.
• Only MM3B explants activated this mechanism since the levels of starch augmented in
salinity, in contrast to what happened in the rest of the mutants and ‘Alemow’.
• However, starch did not seem to play any central role in the regulation of the mutants since
the performance of MM3B did not stand out of the rest of the mutants.

46. Conclusion
• In the saline conditions assayed, all the mutants showed better
productivity than Citrus macrophylla.
• Furthermore, MM5B showed less leaf damage and higher proline levels in
the saline stress conditions than Citrus macrophylla.
• NO3‫־‬ accumulation of was also higher in mutant plants than in Citrus
macrophylla in the saline conditions, which conferred an additional
advantage against stress.

47. Evaluation of MM106 and Omara
apple rootstocks for salt tolerance in vitro
• Objectives: To study the possibility of using in vitro shoot culture in rooting
stage to evaluate salt tolerance of two apple rootstocks (MM106 and Omara).
• Single shoots were cultured on MS medium as a rooting media supplemented
with 0, 20, 40, 60, 80 and 100mM NaCl for 8 weeks.
• Results: All rooting parameters (rooting percentage, roots number, roots
length) and plantlets growth (height of rooted shoot) decreased as salt level
increased in culture medium, with the reductions generally greater for
Omara than MM106 rootstock.
• Also NaCl effect resulted in plantlet necrosis and a reduction in total chlorophyll
content of both rootstocks.
• However, plantlets of MM106 showed less relative root and shoot growth
reduction under salt stress compared with Omara, therefore, it appeared to be
more salt tolerant in vitro than Omara.
Abdel-Hussein (2006)University of Kufa, Iraq

48. • Rooting percentage decreased due to inhibitory effects of salt on the metabolic
activities which associated with cell division, differentiation and elongation and
these processes companied with adventitious root initiation on the base of
microcuttings.
• Endogenous auxin (IAA) and rooting co-factors levels reduced, which might lead to
reduce root initiation, and these substances (especially IAA) play an important
regulatory role in root formation and their development
Effect of NaCl on rooting of MM106 and Omara apple shoots in vitro after 8 weeks

49. Effect of NaCl concentrations on root characters 0f MM106 and Omara apple
rootstocks during rooting stage.

50. • Biosynthesis of chlorophyll was generally inhibited by the depressive effect of stress.
• Under stress conditions, absorption of some ions (Mg and Fe) reduced, which were
involved in the chlorophyll formation.
• It further leads to chlorophyll suppression in leaves or an increase of some growth
inhibitors, such as E or ABA production which enhances senescence.
Efects of NaCl on height and total chlorophyll content of MM106
and Omara apple rootstocks plantlets after 8 weeks in culture

51. The enhancement of tolerance to salt and cold stresses by
accumulating salicylic acid content via the cytosolic malate
dehydrogenase gene in transgenic apple plants
• Objective: To characterize the role of an apple cytosolic malate
dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses
and investigated its regulation mechanism in stress tolerance.
• Material: ‘Gala’ apple in vitro shoot cultures were used for the gene
expression assays, genetic transformation and other analyses. The ‘Gala’
cultures were grown on MS subculture media.
• Three-year old pot-cultured WT and transgenic apple trees were used for salt
treatment.
• Salt stress treatments, that is at 50-mM NaCl for 10 days and subsequent 100-
mM NaCl for 9 days.
• At 8 °C for 7 days and subsequent 0 °C for 7 days in a 14-h light photoperiod.
Wang et al (1997)
Shandong Agricultural University, Tai-An, Shandong, China

52. STRESS TREATMENTS
1. NaCl (19 days): 50-mM NaCl for 10 days and subsequent 100-mM NaCl for 9 days.
2. Cold (14 days): At 8 °C for 7 days and subsequent 0 °C for 7 days in a 14-h light photoperiod.
• Contents of chlorophyll (a + b) of the leaves from the transgenic apple cultures (WT,
Line 5 and Line 7) under normal and stress conditions of NaCl & cold treatment
• Gene expression analysis was done.

53. Conclusion
• The MdcyMDH transcript was induced by mild cold and salt treatments,
and MdcyMDH-overexpressing apple plants possessed improved cold and
salt tolerance compared to wild-type (WT) plants.
• A digital gene expression analysis revealed that MdcyMDH overexpression
largely altered some biological processes, including hormone signal
transduction.
• The transgenic plants produced a high H2O2 content, but a low O2-
production rate was observed compared to the WT plants.
• The transgenic plants accumulated more free and total salicylic acid (SA)
than the WT plants under normal and stress conditions.
• Taken together, MdcyMDH conferred the transgenic apple plants a higher
stress tolerance by producing more ROS and increasing the SA level.
• MdcyMDH could serve as a target gene to genetically engineer salt- and
cold-tolerant trees.

54. SUITABILITY OF DIFFERENT FRUIT CROPS
UNDER
WATER STRESS CONDITIONS

55. Physiological and biochemical responses of young olive
trees (Olea europaea L.) to water stress during flowering
• Objective: To examine physiological and biochemical changes in three Moroccan
varieties (Moroccan Picholine, Menara and Haouzia) of young olive trees (Olea
europaea L.) grown under three different water regimes.
• T0 (severe water stress ): Another 12 pots were not irrigated during the water
treatment period.
• T50 (moderate water stress ): Twelve pots received half the water needed to
maintain the soil at field capacity.
• T100 (control; Well watered plants): Twelve pots of each cultivar were watered
twice a week to field capacity (≈800 mL) and represented the control and well-
watered plants (T100).
• Leaf relative water content (RWC), water potential (Ψw), transpiration rate (E),
stomatal conductance (gs), maximum quantum efficiency of PSII (Fv/Fm), the
contents of total chlorophyll (TCC), proline (ProC) and soluble sugars (SSC) were
measured at the flowering stage during three growing seasons (2015, 2016 and
2017).
Yamani et al (2018)Sidi Mohamed Ben Abdellah University, Morocco

56. Leaf relative water content (RWC), water potential (Ψw), transpiration rate (E), stomatal conductance (gs),
total chlorophyll content (TCC), maximum quantum efficiency of PSII (Fv/Fm), proline content (ProC) and
soluble sugars content (SSC).
• Among the growing seasons, 2015 flowering displayed the lowest values for RWC, Ψw, E,
gs, TCC and Fv/Fm, and the highest for ProC and SSC. (Temperatures was high in 2015. Under
such conditions, olive plants can reduce excessive water loss by closing their stomata and
accumulating osmolytes to facilitate the retention of water and prevent membrane damage).
• Among plant varieties, no significant differences were observed. All are suitable due to high
chlorophyll content and low proline accumulation).
• Correlation studies highlighted significant associations between most parameters.
• Positive relationships were found between RWC, Ψw, E, gs, Fv/Fm and TCC, while all of
these parameters were negatively linked to ProC and SSC.
Severe
Moderate
Control

57. Leaf mechanisms involved in the response of
Cydonia oblonga trees to water stress and recovery
• Objective: To identify the strategy by which quince plants cope with water
stress and to further elucidate the resistance mechanisms developed in
response to water stress and during recovery.
• Old quince trees (cv. BA-29) were subjected to two irrigation treatments:
• Control (T0) plants were drip irrigated (105% ETo) to ensure non limiting
soil water conditions.
• T1 plants were irrigated at the same level as used in T0, except that irrigation
was withheld for 42 days during the linear fruit growth phase (i.e. 209–251
days of the 2016 year ; DOY) , after which irrigation returned to the levels of
T0 (recovery period i.e. 251-266 DOY).
• During the experimental period, T0 and T1 received a total of 374 and
143mm water, respectively, including rain water.
• Leaf water potential (Ψleaf), turgor potential (Ψp leaf) and leaf conductance
(gleaf) values for quince plants was noted.
Miguel Hernández Polytechnic University of Elche, Orihuela (Spain)
Griñán et al (2019)

58. Diurnal course of leaf water potential (Ψleaf), turgor potential (Ψp leaf) and leaf conductance
(gleaf) values for quince plants in T0: CONTROL (closed circles) and T1 (open triangles)
treatments at three different times during the stress period (DOY 224, 238, 251).
Results: From the beginning of water stress to the time of maximum water stress, leaf turgor
was maintained, possibly due to active osmotic adjustment (stress tolerance mechanism).
This leaf turgor maintenance may have contributed to the high leaf conductance, and,
therefore, good leaf productivity.

59. Predawn (Ψpd, A) and midday (Ψmd B) leaf water potential values
Predawn (Ψppd, C) and midday (Ψpmd, D) leaf turgor potential values
T0: CONTROL (closed circles) and T1 (open triangles) treatments

60. Midday stem water potential (Ψstem, A) & midday leaf conductance (glmd, B) values.
T0: CONTROL (closed circles) and T1 (open triangles) treatments
Conclusion: During the development of the severe water stress and recovery
periods, quince trees showed stress tolerance mechanism by active osmotic
adjustment.
This leaf turgor maintenance would allow substantial leaf conductance levels and,
therefore, good leaf productivity.

61. Rootstock-induced molecular responses associated with
drought tolerance in sweet orange as revealed by RNA-Seq
• Objective: To investigate the poorly understood molecular responses underlying
the rootstock-induced drought tolerance in sweet orange.
• RNA-Seq transcriptome analysis was carried out in leaves of sweet orange grafted
on ‘Rangpur’ lime subjected to control and drought-stress treatments.
• A total of 41,827 unique transcripts were identified, among which 1764
transcripts showed significant variation between the treatments, with 1081
genes induced and 683 repressed by drought-stress treatment.
• RNA-Seq data were validated by quantitative real-time PCR (qPCR) analysis.
• The transcripts were distributed in 44 different categories of cellular component,
molecular function and biological process.
• Several genes related to different pathways, were differentially regulated by
drought stress.
• Conclusion: This study provides a useful reference for further exploration of the
functions of candidate genes and applications on the genetic improvement of
citrus rootstocks.
Gonçalves et al (2019)
Universidade Estadual de Santa Cruz, Ilhéus, Brazil

62. Functional categorization of the differentially expressed genes into 44 different categories of
cellular component, molecular function and biological process in leaves of drought-stressed sweet
orange (Citrus sinensis) grafted on ‘Rangpur’ lime.
Categorization was performed using BGI WEGO.

63. A schematic model of the rootstock-induced transcriptional response associated with
drought tolerance in leaves of sweet orange.
Dotted lines represent indirect connections and solid lines represent direct connections.
Abbreviations:
• PKs: protein kinases
• PYR/PYL/RCAR: pyrabactin
resistance 1/PYR1-like/regulatory
components of ABA receptor
• PP2C: protein phosphatase type 2C
• SnRK2: SNF1- related protein
kinase 2
• DREB2A: dehydration-responsive
element binding protein 2A
• HSP: heat shock protein
• MPKs: mitogen-activated kinase
• PRP: proline-rich protein;
• WAKs: wall-associated kinases
• TFs: transcription factors

64. Effect Of Drought Stress In In Vitro And Drought-related
Gene Expression In Carrizo Citrange
• Objective: To investigate the effect of in vitro drought stress on Carrizo
citrange and expression levels of drought related genes.
• Drought stress experiment was established with plants obtained from
germinated seeds of Carrizo citrange.
• For this purpose, MS mediums with 0, 1, 2, 4 and 6% PEG 8000 were
prepared and the plants were transferred to these media.
• 1 mg/L BA was added to promote micropropagation of plants into the MS
medium.
• The plants were sub cultured 3 times at each 4 weeks.
• qRT-PCR Analysis: qRT-PCR analyzes were performed on leaves.
• 15 different plant materials from 3 subcultures were used for the total of 5
concentrations.
• RNA isolation was performed from the leaves. Expression levels of 3 different
genes was noted.
Simsek (2018)
Cukurova University, Adana, Turkey

65. • Decrease in the multiplication coefficient in increasing PEG doses.
• Three genes had accelerating expression levels with increasing PEG doses.
• With these results, it has been revealed that drought-related genes are triggered.

66. 1 SC-PEG 0: Sub culture 1 PEG 0% 1 SC-PEG 6: Sub culture 1 PEG 6%

67. 1 SC-PEG 0: Sub culture 1 PEG 0% 1 SC-PEG 6: Sub culture 1 PEG 6%

68. 1 SC-PEG 0: Sub culture 1 PEG 0% 1 SC-PEG 6: Sub culture 1 PEG 6%

69. Drought Tolerance of Some Wine Grape Cultivars
Under In Vitro Conditions
• Objective: To determine the drought tolerance of 6 different economically important
wine grape cultivars (‘Sultani Seedless’, ‘Çalkarası’, ‘Emir’, ‘Boğazkere’, ‘Öküzgözü’,
‘Narince’) of Turkey under in vitro conditions.
• Drought stress was induced on in vitro-grown explants by 3 different PEG (8000)
(poly ethylene glycol) doses (2, 4 and 6 %).
• Plants were subjected to drought stress for 6 weeks.
• Plant fresh weight, dry weight, shoot length, number of shoots, number of leaves,
electrolyte leakage, relative water content, proline content were determined.
• Being more distinctive at higher doses, PEG treatments yielded significant decreases
in fresh weight, dry weight, shoot length, number of shoots and number of leaves &
increase in proline content.
• Conclusion: Öküzgözü and Sultani Seedless cultivars maximum G & Dvp under
drought stress; seemed to be more resistant to drought.
• But considering the other parameters (Electrolyte leakage, RWC and proline), it is
better to indicate that there were not significant differences in drought tolerance
of the cultivars.
Neval and Rüstem (2019)
Gaziosmanpaşa University, Tasliciftlik, Tokat-Turkey

70. The effects of different PEG doses applied to varieties on growth and development
parameters

71. The effect of different PEG doses applied to the varieties on ion leakage, relative
water content & proline accumulation

72. Genome-wide characterization and expression analysis of
citrus NUCLEAR FACTOR-Y (NF-Y) transcription factors
identified a novel NF-YA gene involved in drought-stress
response and tolerance
• Nuclear factor Y (NF-Y) is a ubiquitous transcription factor found in
eukaryotes. It is composed of three distinct subunits called NF-YA, NF-YB
and NF-YC.
• NF-Ys have been identified as key regulators of multiple pathways in the
control of development and tolerance to biotic and abiotic factors.
• Objectives:
1. To identify and characterize the complete repertoire of genes coding for
NF-Y in citrus.
2. To perform the functional characterization of one of its members, namely
CsNFYA5, in transgenic tobacco plants.
Pereira et al (2018)
Universidade Estadual de Santa Cruz, IlheÂus, Bahia, Brazil

73. Experiment No. 1
• Two-year-old sweet orange (Citrus sinensis L. Osb. var. `Westin') plants grafted
on Rangpur lime (C. limonia Osbeck) were used in the drought stress
experiment.
1. Control (leaf predawn water potential between -0.2 to -0.4 MPa).
2. Drought stress (leaf predawn water potential of -1.5 MPa) treatments.
• RNA extraction and quantitative real-time RT-PCR (qRT-PCR) analysis
• All procedures of RNA extraction, cDNA synthesis and quantitative real-time
RT-PCR (qRT-PCR) analysis were performed.
• Identification and sequence analysis of citrus NF-Ys
• Data obtained from qRT-PCR was compared with sweet orange genome data
available at the Phytozome database.
• Results: A total of 22 genes coding for NF-Y were identified in the genomes of
sweet orange (Citrus sinensis) and Clementine mandarin (C. clementina),
including 6 CsNF-YAs, 11 CsNF-YBs and 5 CsNF-YCs.

74. Experiment No. 2
• The plants of Nicotiana tabacum cv. Havana used in Agrobacterium-mediated
genetic transformation experiments.
• The stress tolerance of wild-type (WT) and transgenic tobacco plants (NF12,
NF15, NF16, NF20 and NF22) was examined by in vitro drought stress tolerance
assay under control and PEG treatments for 30 days.
• CsNF-YA5 expression analysis in response to drought stress
• Overexpression of CsNF-YA5 in transgenic tobacco plants contributed to the
reduction of H2O2 production under dehydration conditions and increased
plant growth and photosynthetic rate under normal conditions and drought stress.
• These biochemical and physiological responses to drought stress promoted by
CsNF-YA5 may confer a productivity advantage in environments with frequent
short-term soil water deficit.

75. In vitro drought stress tolerance assay of CsNFYA5-overexpressing transgenic lines.
Representative phenotypes of control (WT and PC) and CsNFYA5 transgenic (NF12, NF15, NF16,
NF20 and NF22) lines grown under control and PEG treatments for 30 days.
Fig. A Seedling biomass & Fig. B root length.

76. Representative phenotypes of control (WT and PC) and CsNFYA5 transgenic (NF12,
NF15, NF16, NF20 and NF22) lines grown under control and PEG treatments for 30
days.
Fig. C Seedling biomass & Fig. D root length.

77. Physiological and morphological adaptations of the fruit
tree Ziziphus rotundifolia in response to progressive
drought stress
• Objective: To study physiological basis of drought resistance in Ziziphus
rotundifolia Lamk.
• Three irrigation regimes were imposed over a 34-day period:
• Irrigation treatment (control).
• Gradual drought stress treatment (50% of water supplied in the irrigation
treatment i.e. control).
• Rapid drought stress treatment (no irrigation).
• Changes in gas exchange, water relations and solute concentrations of leaves,
stems and roots were determined.
• Results: The differential rate of stress development in the two drought
treatments did not result in markedly different physiological responses, but
merely affected the time at which they were expressed.
• Conclusion: Z. rotundifolia showed a range of responses to different drought
intensities indicating a high degree of plasticity in response to water deficits.
Arndt et al (2001)
Institute of Ecology and Conservation Biology, Vienna, Austria

78. Changes in soil water content in the irrigated, gradual stress and rapid stress treatments.
• In the gradual stress treatment, pot soil water content declined steadily, reaching 26%
of the initial pre-stress value after 34 days.
• In the rapid stress treatment, 76% of the initial water was lost within 16 days. Soil water
content continued to decline throughout the study, reaching 16% of the initial water
content on Day 34.

79. (a) Predawn leaf water potential
(b) Predawn osmotic potential of leaf tissue sap
(b) Stomatal conductance (gleaf)
(c) Intrinsic WUE
The initial response to decreasing soil water content was reduced stomatal conductance,
effectively maintaining predawn leaf water potential (Ψleaf), controlling water loss and
increasing intrinsic water-use efficiency, while optimizing carbon gain during drought.

80. Concentrations of starch, proline & sugars in irrigated and drought-stressed Ziziphus
rotundifolia trees at three harvests.
• As drought developed, osmotic potential at full turgor decreased and total solute
concentrations increased in leaves, indicating osmotic adjustment.
• Decreases in leaf starch concentrations and concomitant increases in hexose sugars and
sucrose suggested a shift in carbon partitioning in favor of soluble carbohydrates.
• In severely drought-stressed leaves, increase in proline concentration, suggests an
osmoprotective role for proline.

81. Physiological, biochemical and molecular responses in
four Prunus rootstocks submitted to drought stress
• Objective: To study physiological, biochemical and molecular drought
responses of four Prunus rootstocks (GF 677, Cadaman, ROOTPAC 20 and
ROOTPAC® R) budded with ‘Catherina’ peach cultivar.
• Two water treatments: well irrigated and water stress.
• Control plants were watered daily and water status was maintained at full
field capacity.
• Water-stressed plants were also irrigated daily, but adding ~80% of the water
evapotranspired the previous day and subjected to progressive water stress
during 26 day.
• Photosynthetic and gas exchange parameters were determined. Root and leaf
soluble sugars and proline content were also measured.
• At the end of the experiment, stressed plants showed lower net photosynthesis
rate, stomatal conductance and transpiration rate, and higher intrinsic leaf
WUE (AN/gs).
• Soluble sugars and proline concentration changes were observed, in both root
and leaf tissues, especially in an advanced state of stress.
Zaragoza, Spain Jiménez et al (2013)

82. Daily soil volumetric water content of control and drought-stressed pots
containing Prunus rootstocks (Cadaman, CD; GF 677, GF; ROOTPAC 20, R20; and
ROOTPAC® R, RR) budded with var. ‘Catherina’.

83. Daily scion apical growth of control and drought-stressed four Prunus
rootstocks budded with var. ‘Catherina’.

84. Midday stem water potential (Ψs) in scion leaves of control and drought-stressed Prunus
rootstocks Cadaman (a), GF 677 (b), ROOTPAC 20 (c) and ROOTPAC® R (d) budded with var.
‘Catherina’.

85. Scion leaf soluble sugars and proline (mg g−1 DW) concentration (n = 6) in control and
drought-stressed Prunus rootstocks after 16 & 26 days of treatment.
The accumulation of proline in roots and leaves with drought stress was related to the
decrease in osmotic potential and increase in WUE
Whereas the accumulation of sorbitol in leaves, raffinose in roots and proline in both
tissues was related only to the increase in the WUE.
16 days
26 days

86. Expression profiles:
• Raffinose synthase (SIP1)
• Δ-1-pyrroline-carboxylate synthase
(P5SC)
• sorbitol 6-phosphate dehydrogenase
(S6PDH) gene
In leaves & roots Cadaman (CD) and GF
677 (GF) rootstocks’ and submitted to
control and drought treatments for 16
days.
• Higher expression of P5SC gene in
roots was also consistent with
proline accumulation in the tolerant
genotype GF 677.
• Accumulation of sorbitol, raffinose
and proline in different tissues
and/or the increase in P5SC
expression could be used as markers
of drought tolerance in peach
cultivars grafted on Prunus
rootstocks.

87. Conclusion
• The biochemical responses to drought, mainly accumulation of sorbitol, raffinose
and proline, were consistently related to the physiological responses to water
stress that confer tolerance.
• Initial molecular responses were related to the biochemical responses observed.
• Accumulation of leaf sorbitol, root raffinose and root and leaf proline could
be implemented as a drought tolerance markers for early selection of Prunus
rootstocks for peach trees under controlled conditions.
• The differential expression of PSC5 in roots could also be used as drought
tolerance marker.
• The almond-based rootstocks GF 677 and ROOTPAC® R showed better
performance to drought stress with both physiological and biochemical
responses.
• The different rootstock performance could be related to their different genetic
background and vigour.
• This study would be the basis for future analysis at the whole-molecular level to
illuminate the tolerance mechanisms to drought in Prunus rootstocks.

88. Tolerance of citrus plants to the combination of high temperature
and drought is associated to the increase in transpiration modulated
by a reduction in abscisic acid levels
• Objective: To study physiological parameters and the hormonal regulation of
Carrizo citrange and Cleopatra mandarin, two citrus genotypes, in response to
the combined action of high temperatures and water deprivation.
• Four experimental groups of each genotype:
• Well-watered plants at 25 °C (CT: control)
• Well-watered plants at 40 °C (HS: heat stress)
• Drought affected plants at 25 °C (WS: water stress)
• Drought affected plants at 40 °C (WS +HS: Combo of both).
• Leaf tissue was sampled at 24 h after subjecting plants to both stresses.
• Results: Carrizo citrange is more tolerant to the stress combination than
Cleopatra mandarin.
Zandalinas et al (2016)
Universitat Jaume I, Castelló de la Plana, Spain

89. Intact sprouts (%) of Carrizo and Cleopatra seedlings subjected to drought
and heat stress (40 °C) in combination for 10 days.

90. Relative water content (RWC) (a) and proline concentration (b) in Carrizo and
Cleopatra plants subjected to drought (WS), heat (HS) and their combination (WS +
HS).

91. SA levels in citrus plants subjected to different stress treatments
• SA accumulated in response to individual stresses but to a higher extent in
samples subjected to the combination of heat and drought (showing an
additive response).
• SA accumulation correlated with the up-regulation of pathogenesis-related
gene 2 (CsPR2), as a downstream response.

92. • ABA accumulation was higher in water-stressed plants followed by that
observed in plants under stress combination.
• ABA signaling in these plants was confirmed by the expression of responsive
to ABA-related gene 18 (CsRAB18).
ABA levels in citrus plants subjected to different stress treatments

93. SUITABILITY OF FRUIT CROPS UNDER
HIGH TEMPERATURE STRESS
CONDITIONS

94. High temperatures change the perspective: integrating
hormonal responses in citrus plants under co-occurring
abiotic stress conditions
• Objective: To study physiological, hormonal and molecular responses of the citrus rootstock,
Carrizo citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L. Osb.) subjected to wounding or
high salinity occurring individually or in combination with heat stress.
• Six experimental groups were established:
1. Intact plants growing at 25 ᵒC (control, CT)
2. Intact plants growing at 40 ᵒC (heat stress, HS)
3. Wounded plants growing at 25 ᵒC (wounded, Wo Wounding was performed by drilling 50%
of the leaves with a paper puncher )
4. Wounded plants growing at 40 ᵒC (wounded + heat shock, Wo+HS),
5. Salinized plants growing at 25 ᵒC (NaCl 60mM NaCl)
6. Salinized plants growing at 40 ᵒC (NaCl+HS).
• All conditions were applied at the same time for 15 days.
• Conclusion: Co-occurring abiotic stress conditions can modify (either enhance or reduce) the
hormonal response to modulate specific responses.
Balfagón et al (2018)
Universitat Jaume I, Castelló de la Plana, Spain

95. Leaf RWC of Carrizo plants subjected to wounding (Wo), salt stress (NaCl), heat
stress (HS), combination of wounding and heat stress (Wo+HS) and combination
of salt and heat stress (NaCl+HS) for 15 days.

96. Content of ABA, JA & SA of Carrizo plants subjected to wounding (Wo), salt
stress (NaCl), heat stress (HS), combination of wounding and heat stress
(Wo+HS) and combination of salt and heat stress (NaCl+HS) for 15 days.

97. Relative expression
of genes involved in
ABA biosynthesis
CsNCED1
CsAAO3
Signaling
CsAREB1
CsAREB2
CsRD22
CsRD29

98. Relative expression
of genes involved in
JA biosynthesis
CsLOX
CsAOS
CsJAR1
Signaling
CsVSP2

99. Relative expression of
genes involved in
SA biosynthesis
CsICS1
CsPAL
Signaling
CsPR2

100. Involvement of ascorbate peroxidase and heat shock
proteins on citrus tolerance to combined conditions of
drought and high temperatures
• Objective: To study stress tolerance mechanism in two citrus rootstocks
Carrizo citrange (Poncirus trifoliata L. Raf. X Citrus sinensis L. Osb.) and
Cleopatra mandarin (Citrus reshni Hort. ex Tan.) with contrasting tolerance to
drought and heat stress.
• Four experimental groups of each genotype:
• Well-watered plants at 25 °C (CT: control)
• Well-watered plants at 40 °C (HS: heat stress)
• Drought affected plants at 25 °C (WS: water stress)
• Drought affected plants at 40 °C (WS +HS: Combo of both).
• Leaf tissue was sampled 24 h after the stress imposition.
• Results: Carrizo citrange is more tolerant to the stress combination than
Cleopatra mandarin.
Zandalinas et al (2016)
Universitat Jaume I, Castelló de la Plana, Spain

101. Stress-induced leaf damage
Carrizo citrange (A, C and E)
Cleopatra mandarin (B, D and F)
A and B: Intact leaves
C and D: Chlorotic leaves
E and F: Partially necrotic leaves.

102. Leaf damage & stomatal conductance (gs) in Carrizo and Cleopatra plants
subjected to different stress
Less leaf damage in Carrizo than in Cleopatra under combined conditions of drought and
heat stress.
Stomatal conductance increased in heat stress due to HSP.

103. APX activity and APX2 gene expression in Carrizo and Cleopatra plants.
• APX activity was less under water stress conditions, moderate under WS+HS conditions, but
highest under HS conditons.
• And it was comparatively higher in Carrizo than in Cleopatra under stress combination.
• Higher APX2 gene expression in Carrizo, contributes to the higher tolerance of Carrizo plants
to the combination of stresses.
• Carrizo is better rootstock than Cleopatra (traditionally used in areas with scare water supplies).

104. APX2, HSP101 and HSP17.6 levels in Carrizo and Cleopatra plants subjected to stress.
Top: Quantification graphs
Bottom: Representative protein blots and loading controls

105. Conclusion
• The pattern of accumulation of three proteins (APX, HSP101 and HSP17.6)
involved in abiotic stress tolerance shows that they do not accumulate under
water stress conditions individually applied.
• Contents of APX and HSP101 are higher in Carrizo than in Cleopatra under
stress combination
• Whereas HSP17.6 has a similar behavior in both types of plants.
• This, together with a better stomatal control and a higher APX activity of
Carrizo, contributes to the higher tolerance of Carrizo plants to the combination
of stresses.
• Carrizo is better rootstock than Cleopatra (traditionally used in areas with scare
water supplies) under the predictable future climatic conditions with frequent
periods of drought combined with high temperatures.
• This work also provides the basis for testing the tolerance of different citrus
varieties grafted on these rootstocks and growing under different field
conditions.

106. Effect of high temperature stress on the reproductive growth
of strawberry cvs. ‘Nyoho’ and ‘Toyonoka’
• Objective: To examined the effect of two day/night temperature regimes on fruit
set and fruit growth in two cultivars, ‘Nyoho’ and ‘Toyonoka’.
• 10 plants of each cultivar were two growth chambers kept at either 23/18 C
(control) or 30/25 C (high) day/night temperatures.
• The percentage of fruit set in ‘Nyoho’ was not significantly different between the
two temperature treatments, while that in ‘Toyonoka’ was much lower at 30/25 C
than at 23/18 C.
• Days to ripening was shorter at 30/25 C than at 23/18 C, and no cultivar
differences were observed.
• Fresh weight & diameter of fruits reduced at 30/25 8C in both cultivars
• Percentage of fruit set at 30/25 C was also significantly lower in ‘Toyonoka’ than in
‘Nyoho’. ‘Nyoho showed resistance to high temperature.
• These results indicate that high temperature stress negatively affects the
reproductive process in strawberry and that plant response to high temperature
stress is cultivar specific too.
Ledesma et al (2008)University of Tokyo, Japan

107. Fully ripe fruits obtained from ‘Nyoho’ (A) and ‘Toyonoka’ (B) strawberry plants
grown at 30/25 C.
In ‘Toyonoka’, low fruit set caused malformations and reductions in the sizes of fruits.
Mean number of inflorescences Fresh weight of fruits

108. A Study on the High Temperature Treatment to Three
Grapevine Varieties Grown Under In vitro Conditions
• Objective: To determine temperature grades and exposure times to stresses
in plants of vine varieties grown in vitro conditions.
• Three varieties: Çalkarası, Öküzgözü and Narince).
• The durations of the stress conditions were determined with the preliminary
study carried out with the Çalkarası variety.
• Then, the three varieties used in the study were exposed to stress for the same
durations.
• Plants with appropriate sizes (5-6 leaves) were placed in a plant growth
cabinet at 25ºC with a photoperiod of 16 h.
• Heat stress at temperatures of 35ºC and 40ºC were applied.
Altıncı et al (2018)
Gaziosmanpaşa University Tokat, TURKEY

109. In vitro plants of Çalkarası exposed to 35 ºC stress at different times (24, 36, 48 hours)
In vitro plants of Çalkarası exposed to 40 ºC stress at different times (9, 12, 15 hours)
Exposure time 36 hours at 35˚C and 12 hours at 40˚C did not lose the vitality of the plants, but
plants seriously entered the stress.

110. 35 ºC stress application for 36 hours in Çalkarası, Öküzgözü and Narince
40 ºC stress application for 12 hours in Çalkarası, Öküzgözü and Narince
More damage was found at 40 ºC than 40 ºC stress application.
Among three varieties, least damage was found in Öküzgözü.

111. Climate warming enhances flower formation, earliness of
blooming and fruit size in plum (Prunus domestica L.) in the cool
Nordic environment
• Objective: To analyse data from a 31-year series of observations (1985–2016) on
spring phenology and flowering and fruiting performance of three plum cultivars in
an experimental orchard at Ås in southeast Norway (59° 40′N; 10° 50′E).
• As temperature increased in March and April, the date of full bloom (FB) reduced
than earlier low temp. On average for all cultivars, blooming was advanced by 10
days over the study period.
• August and September temperature, which also increased significantly during the
study period, was closely positively correlated with the amount of flowering in the
subsequent spring and also interacted with early spring temperature in
advancing blooming time.
• Conclusion: Increasing March and April temperatures during the last 30 years has
advanced blooming and spring phenology in plum and the resulting extension of the
growing season has led to increasing fruit size at harvest.
• Climate warming appears to have been positive for plum production in the cool
Nordic environment.
Woznickia et al (2019)
Norwegian University of Life Sciences, Ås, Norway

112. Trends in March, April and May temperatures at Norway, and the corresponding
trends in FB from 1 January for the three studied plum cultivars over the years
1985–2016.

113. Scatterplot and trend line for the mean fruit size at harvest for each year
across the 1985–2016 period for the three studied plum cultivars.

114. Late blooming was negatively correlated with fruit size at harvest for all cultivars.
Early blooming had significant positive correlation between fruit size at harvest and the
number of days from FB to harvest for all cultivars.
Since the period between blooming and fruit maturity (harvest) increased during the observed 31-
year period, fruit size at harvest also increased across the years for all cultivars.

115. SUITABILITY OF DIFFERENT FRUIT CROPS
UNDER
LOW TEMPERTURE STRESS CONDITIONS

116. Evaluation of susceptibility of pear and plum
cultivars to winter frost
• Objective: To investigate winter frost damage to pear and plum buds in
Mashhad, after a period of unusually warm weather for a few days in March 2013
followed by a sudden drop in temperature to -12°C.
• Two separate experiments included.
• Twelve cultivars of pear: William’s, Bell de june, Ida, Spadona, Koshia, Domkaj,
Dare Gazi, Mohamad Ali, Boheme, Asian pear, Shekari and Ghodumi.
• Eight plum cultivars (European: Prunus domestica L. Stanley, Early Santa Rosa,
Late Santa Rosa, Shablon, Black Diamond; Japanese: P. salicina L. Shiro, Shams,
Computi)
• Frost damages of vegetative and reproductive buds and spur-part below the buds
of pear and plum cultivars were investigated based on visual observations.
• After 24 h keeping at laboratory temperature, a hundred vegetative and
reproductive bud samples of each cultivar were visually evaluated for brown
discoloration.
Khorshidi et al (2014)
Ferdowsi University Of Mashhad, Mashhad, Iran

117. • Minimum & maximum temperatures from February 19th to March 20th, 2013.
• For most of the time the lowest temperature was not below zero in February and
March until March 7th, when it reached -5.2°C, and the lowest one was -12.2°C on
March 9th.

118. Vegetative bud of pear var. ‘Ghodumi’ suffered the most damage.
Reproductive bud of ‘Ghodumi’ suffered the least damage.
The EC and proline content of ‘Dare Gazi’ were the highest despite it did not show
high resistance. (Reason: Proline increase doesn’t increase cold resistance).

119. Percentage of frost damage of pistil and male organs of some plum cultivars
• In all plum cultivars the pistil suffered more damage than the male part.
• Percentage of cold injury in flower bud was higher than other tissues, and the
cold hardiness of pistil was weaker than stamen.

120. • All parts of ‘Late Santa Rosa’ were the most susceptible in plum cultivars.
• ‘Computi’ had the most resistant reproductive bud and no damage was observed
in the other parts. It had the lowest proline content.
• Conclusion: Proline index is not appropriate for evaluating frost damage, but
there was a high correlation was between EC and frost damage.

121. Metabolic Responses to Low Temperature of Three Peach
Fruit Cultivars Differently Sensitive to Cold Storage
• Refrigerated storage is widely applied in order to maintain peach quality but it can also
induce chilling injuries (CIs) such as flesh browning and bleeding.
• Objective: To study changes in both metabolome and volatilome induced by cold
storage in the mesocarp of three peach cultivars (‘Red Haven’, RH, ‘Regina di Londa’, RL,
and ‘Flaminia’, FL).
• Fruits of ‘Red Haven’ (RH, yellow fleshed), ‘Flaminia’ (FL, yellow-fleshed, C35 days
from RH harvest), ‘Regina di Londa’ (RL, white fleshed, C55 days from RH harvest)
were harvested at flesh firmness values of 37, 42, and 56 N for RH, FL, and RL,
respectively,
• Immediately after transfer to the lab, fruit were incubated in cold chambers at two low
temperature conditions (0.5 and 5.5 C) and reference (control) at 20 C.
• Peaches were sampled at harvest (T0) and after 1, 2, 3, and 4 weeks of cold storage,
whereas for the control at 20C the 4th week sampling was not performed due to
over-ripening and decay.
• Considering that CI symptoms appear after fruit removal from cold storage, fruit were
additionally kept for 3 days at room temperature to evaluate the post-storage
behavior and the shelf-life (SL).
Brizzolara et al (2018)
Istituto di Scienze della Vita, Scuola Superiore Sant’Anna, Pisa, Italy,

122. Flesh firmness (A) & TSS (B) measured at end of 3 week in control & at 4 week in cold treatment.
Further cold storage fruits kept for three additional days under shelf-life conditions (20 C,
Shelf Life; dotted lines) and on peaches sampled immediately after cold storage (no SL; solid lines).
Black diamonds: Control fruit 20 C Blue circles: 0.5 C Red squares: 5.5 C
RL showed highest firmness at 0.5 C.
No SL (solid lines) had more firmness than 20 C SL (dotted lines).
FL & RH showed highest TSS at 5.5 C. But RL showed highest TSS at 0.5 C.
20 C SL (dotted lines) showed highest TSS than No SL (solid lines)..
Compared to RL and FL, RH showed a marked less reduction in firmness & TSS

123. Incidence of CIs (flesh bleeding, A & flesh browning, B) measured at end of 3 week in control &
at 4 week in cold treatment. Further cold storage fruits kept for three additional days under shelf-
life conditions (20 C, Shelf Life; dotted lines) and on peaches sampled immediately after cold
storage (no SL; solid lines).
Black diamonds: Control fruit 20 C Blue circles: 0.5 C Red squares: 5.5 C
Flesh bleeding revealed high incidence following 0.5C storage.
Flesh browning appeared to be more related to 5.5C storage.
Compared to RL and FL, RH showed a marked lower incidence of CIs.

124. Conclusion
• RH peaches indeed differ from RL and FL in particular when
considering data from samples collected at the end of the cold storage.
• In all three cultivars, glucose-6 P, xylose, sorbitol, epicatechin, catechin,
and putrescine markedly increased during cold storage, while glucuronic,
mucic and shikimic acids decreased.
• Among volatile organic compounds (VOCs), aldehydes and alcohols
generally accumulated more under low temperature conditions while
esters and lactones evolved during subsequent shelf life.
• The lower levels of flesh browning and bleeding displayed by RH
peaches were related to compounds with antioxidant activity, or acting as
osmotic protectants and membrane stabilizer.
• Indeed, RH showed higher levels of amino acids and urea, together
with a marked increase in putrescine, sorbitol, maltitol, myoinositol and
sucrose detected during storage and SL.

125. Cold Hardiness and Options for the Freeze Protection
of Southern Highbush Blueberry
• Southern highbush blueberries are a low chill species of blueberry that are
commercially grown in sub-tropical climates.
• Due to the nature of SHB, the flowering and fruit set occur in mid-winter to early
spring and are susceptible to freeze damage.
• Of the various methods used in frost protection, applying overhead irrigation
water is the most promising.
• During a freeze event, an application of 6.3 mm/ha of water per hour is required
to protect blueberries from 2.8 C (27 F) temperature with winds from 0 to 16
km/h (0 to 10 miles/h).
• This is 25.4 kL/h/ha of water.
• This paper will review methods of freeze/frost protection, importance of weather
patterns, and critical temperatures based on phenology of flowering to fruit.
• Conclusion: Understanding weather patterns, flower phenology, and the capacity
of the freeze protection system are the basis for mitigating freeze damage to
southern highbush blueberry.
Smith(2019)
University of Georgia, Tifton Campus, Tifton, USA

126. Tight bud (stage 1) Bud swell (stage 2) Bud break (stage 3)
Tight bud and bud swell can tolerate cold temperatures of −12 to −9 °C (10–15 °F).
Bud break is more sensitive to cold, where at −7 °C (20 °F), damage can occur.

127. Tight cluster (stage 4) Early pink bud (stage 5)
Tight cluster can tolerates cold to −7 to −5 °C (20–23 °F).
Early pink bud can tolerate cold to −4.4 to −2.8 °C (23–27 °F).

128. Pink buds (stage 6) Late pink bud (stage 7)
Pink buds are cold tolerant to −5 to −4 °C (23–25 °F).
Late pink bud are cold tolerant to −4.4 to −2.8 °C (24–27 °F).

129. Full bloom (stage 8) Petal fall (stage 9)
Full bloom are cold tolerant to −2.2 °C (28 °F).
Petal fall are cold tolerant 0 °C (32 °F).
Cold tolerance decreased as growth proceed from tight bud to petal fall.
Once the petals fall (stage 9), freeze damage will occur at 0 °C.
Even small green fruit is sensitive to 0 °C.

130. Comparative metabolic profiling of Vitis amurensis and
Vitis vinifera during cold acclimation
• Objective: To investigate accumulation of metabolites in V. amurensis during cold
acclimation (CA) which is wild Vitis plant that can withstand extreme cold temperatures
• Plantlets of V. amurensis and V. vinifera cv. Muscat of Hamburg were treated at 4 °C for
24 and 72 h, and changes of metabolites in leaves were detected.
• Most of the identified metabolites, including carbohydrates, amino acids, and organic acids,
accumulated in the two types of grape after CA.
• Sugars: Galactinol, raffinose, fructose, mannose, glycine, and ascorbate were continuously
induced by cold in V. amurensis, but not in Muscat of Hamburg.
• Twelve metabolites increased in V. amurensis during CA.
• More galactinol, ascorbate, 2-oxoglutarate, and putrescine, accumulated in V. amurensis, but
not in Muscat of Hamburg, during CA.
• All these metabolites are responsible for the excellent cold tolerance in V. amurensis.
• The expression levels of the genes encoding β-amylase (BAMY), galactinol synthase
(GolS), and raffinose synthase (RafS) were evaluated by qRT-PCR.
• In conclusion, these results show the inherent differences in metabolites between V.
amurensis and V. vinifera under CA.
Chai et al (2019)Chinese Academy of Sciences, Wuhan, P.R. China

131. Metabolites that specifically accumulated in V. amurensis and V. vinifera cv. Muscat Hamburg
under the cold stress condition.

132. qRT-PCR results for BAMY gene family members in V. amurensisand V. vinifera cv. Muscat Hamburg.
The expression BAMY (VIT_02s0012 g00170) was primarily responsible
for the accumulation of maltose.

133. qRT-PCR results for GolS gene family members in V. amurensisand V. vinifera cv. Muscat Hamburg.
The expression GoIS (VIT_14s0060g00760) was primarily responsible
for the accumulation of galactinol.

134. qRT-PCR results for RafS gene family members in V. amurensisand V. vinifera cv. Muscat Hamburg.
The expression RafS (VIT_05s0077 g00840) was primarily responsible
for the accumulation of raffinose.

135. ADAPTATION OF DIFFERENT FRUIT CROPS
TO
BIOTIC STRESS

136. Resistance gene analogues in mango against mango malformation
• Plant resistant gene analouges (RGA): Resistant gene having conserved domains
that plays specific roles in pathogens resistance. These are receptors like proteins.
• H-8-11 (A×LS) and Bhadauran showed a more than 50% sequence
similarity to other resistance gene analogues.
• So far malformation has not been observed in these two cultivars.
• These sequences can be used for further studies in the mango malformation
resistance breeding programs.
• The cloning of resistance gene sequences provides the possibility of using
the transgenic approach to increase disease resistance of plants
Ebrahim et al (2013)Indian Agricultural Research Institute, New Delhi, India

137. Evaluation of Different Papaya Cultivars for Yield, Quality
and Papaya Ring Spot Disease under Pune Conditions
• Objective: To access the suitability of different papaya cultivars to Pune
conditions.
• Nine papaya cultivars (Washington, CO 2, CO 6, Pusa Dwarf, Pusa Giant, Pusa
Nanha, Pusa Delicious, Pusa Majesty and Red Lady) were evaluated for
growth, yield, quality and PRSV incidence.
• Results: Cv. Pusa Giant recorded significantly maximum fruit yield/plant
(66.1kg)
• Cv. Red Lady showed early flowering (70.4 Days after planting; DAP) which
was at par with cv. CO 2 (73.5 DAP), Pusa Nanha (73.7 DAP) and Pusa Dwarf
(73.6 DAP).
• Regarding quality parameters viz. pulp thickness and TSS were maximum
(3.1cm and 11.71 o Brix, respectively) in cv. Red Lady
Chalak et al (2016)
College of Agriculture, Pune-411005, India.

138. Evaluation of Papaya Cultivars

139. Reaction to papaya ring spot disease
(PRS)
Among these cultivars, the magnitude of
infestation was lowest DPI (66.0 as
Percentage Disease Index) in Pusa Giant
at 10 months after planting.
The maximum infestation was
observed in widely adopted cv Red Lady
(98.7) at the same age.
From present investigation cv Pusa
Giant was found as a good yielder and
also tolerant to ring spot disease among
these papaya cultivars.

140. Resistance of transgenic papaya plants to
Papaya ringspot virus
• Objective: To generate transgenic papayas resistant against Papaya ringspot virus,
• The full-length coat protein (CP) gene was amplified from the RNA of PRSV
isolated from Ratchaburi province, Thailand by RT-PCR.
• A binary vector containing the coat protein gene under the control of a 35S
promoter, was constructed and transformed into somatic embryos of papaya
cultivar Khak Dum by microprojectile bombardment.
• Eight transgenic lines were identified.
• Integration of the transferred genes into papaya calli was verified by PCR
amplification of the coat protein gene,
• Although the coat protein gene was detected in all transgenic lines, only line G2
was found to be highly resistant to virus. Because coat protein gene was
expressed by this line.
Kertbundit et al (2007)
Mahidol University, Bangkok, Thailand

141. ‘HoneySweet’ (C5), the First Genetically Engineered Plum pox
virus–resistant Plum (Prunus domestica L.) Cultivar
• ‘HoneySweet’ originated as a seedling from the open pollination of ‘Bluebyrd’
plum (Scorza and Fogle, 1999). The pollen parent of ‘HoneySweet’ is unknown.
• ‘HoneySweet’ was originally selected in vitro shoots regenerated from a hypocotyl
slice of Bluebyrd plum seedling.
• Transformed with Agrobacterium tumefaciens carrying the plasmid PPV-CP-
33 (some prunus sp) Transgenic shoots, coded as C5 along with other
transgenic shoots, was rooted in vitro and transferred to a greenhouse. .
• Asexually propagated by bud grafting on different rootstock of plum and peach.
• Aphid inoculations with the different strains of Plum pox virus (PPV Testing
(PCR, ELISA).
• Field evaluation of transgenic plants for almost twelve years in aphid vectored
conditions in different countries later known as Honey Sweet (E).
U.S Kearneysville Scorza et al (2016)

142. Three-year old ‘HoneySweet’ tree in Bistrita, Romania (left), with a weighed
sample of ‘HoneySweet’ fruit (right).

143. Transgenic Virus Resistant Papaya: From Hope to Reality
for Controlling Papaya Ringspot Virus in Hawaii
• Papaya ringspot is the most important disease limiting the production of
papaya worldwide.
• Symptoms of PRSV on Papaya tree (a) and fruit (b)
• Symptoms: Severe leaf mosaic, reduction of leaf canopy as the disease
progresses, stunting of the plant, water-soaked oily streaks on the petiole and
upper part of the trunk, and ringed spots on the surface of the fruits.
Gonsalves et al (2004)U.S. Pacific Basin Agricultural Research Center, Hawaii

144. Effect of rapid virus spread through commercial fields.
A healthy commercial papaya field in Puna in 1940.
Viral epidemic in 1992 & all plants got infected.
PRSV was first reported in Hawaii in the mid-1940s, it did not become a major
problem to the Hawaiian papaya industry until the 1950s (Gonsalves, 2004).

145. How transgenic papaya formed ?
• Isolation of coat protein (CP) gene of the mild mutant PRSV HA 5-1.
• Insertion of coat protein gene in vector pGA482GG
• Resulting plasmid DNA delivered via high velocity micro-projectiles
gun into the papaya cells (Sunset and Kapoho)
• As a result, sufficient number of transgenic papaya plants were obtained.
• R0 micropropagated plants, designated as 55-1, when tested showed
excellent resistance against PRSV.
• Furthur, Line 55-1 was crossed with non-transgenic Sunset under
green house condition with 50 % progenies being transgenic to produce
R1 plants.
• The homozygous line 55-1 named as ‘SunUp’, was red fleshed
transgenic cultivar, the dominant cultivar growing in Hawaii.
• The cross of transgenic SunUp and non-transgenic Kapoho was made
to obtain the yellow fleshed hybrid ‘Rainbow’.

146. • Kapoho field trial started in 1995, showing a solid block of PRSV-resistant
Rainbow growing well while the surrounding susceptible non-transgenic
Sunrise is severely infected with PRSV.
• Picture taken 19 months after start of the field trial.

147. Transgenic papaya line
55-1 showing resistance
to PRSV HA compared to
infected non-transgenic
papaya.

148. Conclusion & Future prospects
• The understanding of the underlying mechanisms of response and the
existing interactions among abiotic & biotic stressors will provide
valuable information for crop improvement.
• Breeding for resistance to exposure to combined abiotic and biotic stress
is needed to be done. It can be done either by incorporation of genetic
components regulating the response or by gene pyramiding to both
stresses.
• Gene cloning: Resistance genes to particular stress should be cloned. The
cloning of resistance gene sequences provides the possibility of using the
transgenic approach to increase stress resistance in plants without
the transfer of undesirable characters to the offspring.

149. THANK
YOU