This document discusses brassinosteroids (BRs), which are plant steroid hormones. It covers the biosynthesis, translocation, functions, and mechanisms of action of BRs. Specifically, it discusses: 1) The isolation of BRs from rape plants and identification of the active component brassinolide. BRs are derived from the triterpene squalene and their precursors include campesterol. 2) BRs stimulate growth, cell division, seed germination, leaf development, and stress responses. They are involved in BR sensing and signaling pathways. 3) The biosynthesis of BRs involves parallel early and late C-6 oxidation pathways that convert campesterol precursors to the

1. Biosynthesis, translocation, physiological role in cell level, basic functions and mechanism
of action of brassinosteroids AND MORPHACTINS
BY
B. PAVAN KUMAR NAIK
DEPT. OF HORTICULTURE
FACULTY OF AGRICULTURE
ANNAMALAI UNIVERSITY

2. INTRODUCTION OF BR
• Isolated from pollen of the rape
plant (Brassica napus), the
active substances were referred
to collectively as brassins.
• M. D. Grove and his coworkers
identified the active component
as brassinolide (BL) steroid
hormones with a chemical
structure similar to the steroid
hormones in animals.
• Brassinosteroids are
polyhydroxylated sterols
derived from the triterpene
(squalene). The precursor to
brassinolide is campesterol, a
C28 sterol.

3. Conti…
• More than 40 analogs of BL isolated
from more than 60 plant, seeds,
leaves, stem, root and flowers. BL at
a conc. Of 10-7 M is able to stimulate
a fourfold increases in the length of
soybean epicotyls.
• Brassinosteroids sensing involves a
serine/threonine kinase that regulates
the phosphorylation and
dephosphorylation of transcription
factors.
• Pollen and immature seeds are the
richest sources of BS with a range of
1–100 μg kg−1 (fresh tissue),
whereas shoots and leaves usually
possess lower amounts of 0.01–0.1
μg kg−1 (fresh tissue).

4. THERAPEUTIC ROLE OF BRS
• Anticancerous,
• antiproliferative,
• antiangiogenesis,
• antiviral,
• antigenotoxic,
• antifungal, and
• antibacterial, and display anti-chloesterolemic
activities, synthetic medication.
Malikova et al. 2008

5. Key breakthroughs in the timeline of
research on brassinosteroids

6. Brassinosteroids-Structure

7. FUNCTIONS OF BR
• Brassinosteroids elicit an
impressive array of developmental
responses, including an increased
rate of stem and pollen tube
elongation, increased rates of cell
division (in the presence of auxin
and cytokinin), seed germination,
leaf morphogenesis, apical
dominance, inhibition of root
elongation, vascular differentiation,
accelerated senescence and cell
death.
• BR are also implicated in mediating
responses to both abiotic and biotic
stress, including salt, drought,
temperature extremes and
pathogens.

8. BIOSYNTHESIS OF BRs
• BRs are C27, C28, and C29
steroids depending on their
C-24 alkyl substituents. At
the time of writing, more
than 50 free, naturally
occurring BRs have been
identified in plants.
• BL (Figure 1) is the most
biologically active C28 BR
and, together with its C28
congeners, is distributed
widely in the plant kingdom.

9. Conti…
• The biosynthetic pathways of BL were initially elucidated using
cultured Catharanthus roseus cells. Extensive metabolic studies
suggested parallel branched BL pathways, namely the early and late
C-6 oxidation pathways.
• Recent studies suggest that there is crosstalk between these parallel
pathways, implying that they are not totally autonomous. The
operation of an early C-22 oxidation branch of the BR biosynthetic
pathway has also been demonstrated.
• Thus, BR biosynthetic pathways are highly networked (Figure 2)
Furthermore, the biosynthesis of C27 BRs has been investigated.
• Recent studies have utilized BR biosynthesis inhibitors. The
identification and regulation of the various enzymes and genes
involved in BR biosynthesis have been subjected to intensive study
by genetic, molecular, and biochemical analyses of dwarf
mutants defective in BR biosynthesis or perception.

10. Early and Late C-6 Oxidation Pathways
• As shown in Figure 2, campesterol is first converted to campestanol, which in turn
is converted to castasterone (CS) through either the early C-6 oxidation or the late
C-6 oxidation. Finally, CS is converted to BL.
• However, some of the steps involved have only recently been clarified. One such
step, the conversion of 6-oxocampestanol to cathasterone, was demonstrated in
cultured C. roseus cells, fully substantiating the pathway from campestanol to BL
through early C-6 oxidation.
• The campestanol → 6-deoxocathasterone → 6-deoxoteasterone sequence in the late
C-6 oxidation pathway has also been established in Arabidopsis, completing the
elucidation of the late C-6 oxidation pathway.
• The biosynthesis of BRs in plants other than Arabidopsis and C. roseus has rarely
been investigated.
• The pathways from teasterone to CS, and from 6-deoxoCS to CS, were
demonstrated in rice and tobacco seedlings.
• In addition, the conversions between teasterone and typhasterol via 3-
dehydroteasterone has been confirmed in cultured cells and the cell-free system of
Marchantia polymorpha and lily cells (1), while the same reactions have been
shown to occur when 24-epimers were fed to cultured cells of tomato.

11. Early C-22 Oxidation Branch in the BR Biosynthetic
Pathway
• Fujioka et al. 2002, identified many novel 22-hydroxylated
C27 and C28 BRs in cultured C. roseus cells and
Arabidopsis seedlings and in parallel metabolic studies
elucidated a new subpathway:
• (22S)-22-hydroxycampesterol → (22S,24R)- 22-
hydroxyergost-4-en-3-one → (22S,24R)-22-hydroxy-5α-
ergostan-3-one → 6- deoxocathasterone.
• These experiments also indicated that the det2 mutant is
defective in the conversion of (22S,24R)-22-hydroxyergost-
4-en-3-one → (22S,24R)-22-hydroxy-5α-ergostan-3-one.
• These findings confirm that BR biosynthesis pathways are
highly networked.

12. -Fujioka and Yokota. 2003

13. Enzymes, Genes, and Related Mutants
• Many of the genes encoding BR biosynthetic enzymes have been cloned using BR
biosynthesis mutants of Arabidopsis, pea, tomato, and rice.
• These mutants are BR deficient and revert to a wild-type phenotype following
treatment with exogenous BRs. Common features of BR mutants are a short robust
stature, short and round dark-green leaves, and reduced fertility or sterility.
• The short and round leaves are ascribed to suppressed cell expansion and
proliferation.
• BR mutants show prolonged development, and exhibit varying degrees of de-
etiolation when grown in the dark.
• Most Arabidopsis BR mutants are de-etiolated in the dark with short hypocotyls and
open cotyledons.
• When grown in the light, these mutants are dwarfs with dark-green, curled
leaves; reduced apical dominance; reduced fertility; and delayed senescence.
• Pea BR biosynthesis mutants are dwarf, but unlike Arabidopsis, they do not have
curled leaves or dark-grown phenotypes.

14. The current status of BR biosynthesis enzymes that
have been cloned is summarized as follows:
SAX1
• The sax1 (hypersensitive to abscisic acid and auxin) mutant was originally isolated as an auxin-hypersensitive
mutant, but it turned out to have striking ABA hypersensitivity.
DET2 (DWF6, CRO1)
• The Arabidopsis det2 mutant was isolated as a de-etiolated mutant involved in light-regulated development.
• Later, dwf6 and cro1 were found to be allelic to det2. The DET2 gene maps to the bottom of chromosome 2, and
encodes a protein similar to mammalian steroid 5α-reductase. DET2 protein, when expressed in human kidney
cells, catalyzes the reduction of several animal steroids, whereas human steroid 5α-reductase rescues det2 mutant
phenotypes.
DWF4 (CRO3)
• The DWF4 gene maps to the lower arm of chromosome 3 and encodes a cytochrome P450 monooxygenase
(CYP90B1) that shares 43% homology with CPD, a putative 23α-hydroxylase.
• In transgenic Arabidopsis plants ectopically overexpressing DWF4, an increase in both hypocotyl and
inflorescence height, prolonged flowering, and enhanced seed yield were observed and the level of 22-
hydroxylated BRs was increased.
CPD (CBB3, DWF3)
• The cpd (constitutive and photomorphogenesis and dwarfism) mutant was isolated using a T-DNA tagging strategy.
This mutant displays deetiolation and derepression of light-induced genes in the dark. In the light, cpd plant
height is 3% to 5% that of wild type.
PEA LK
• The pea lk is an extreme dwarf that is BR deficient because of loss of 5α-reductase activity and therefore is an
ortholog of Arabidopsis det2.

15. Conti…
TOMATO DWARF (D) AND DPY
• The tomato Dwarf gene, isolated by transposon tagging, encodes a
cytochrome P450 (CYP85A1). The extreme dwarf mutant
accumulates 6-deoxoCS and the functional expression of Dwarf in
yeast established that it catalyzes the two-step oxidation of 6-
deoxoCS to CS.
RICE DWARF (OsDWARF)
• In monocots, only BR-insensitive rice mutants have been identified.
• Recently, BR-deficient dwarf1 and BR-dependent dwarf1, both
abbreviated as brd1, have been isolated as mutants with a lesion in
the OsDwarf gene, a tomato Dwarf ortholog.
• These mutations show a range of growth abnormalities, especially
in leaf and stem cell elongation, as well as constitutive
photomorphogenesis.

16. YCZ-18 Is a New Brassinosteroid Biosynthesis Inhibitor
• Biological activities of YCZ-18 in Arabidopsis under different
growth conditions.
• three complementary growth conditions were assigned to ensure the
capture of data describing the impact of YCZ-18 to Arabidopsis
growth and development over the entire life of the plants.

17. Conti…
1. The first growth condition, growth on plates for a period of 1–2
weeks, demonstrates the effects of YCZ-18 on early seedling
growth.
2. The second condition is a hydroponic growth condition that has
been validated for studies on root development.
3. The third condition consists of spraying the chemicals on the
plants grown in soil for a period of approximately 2 months.
RESEARCH
STUDY

18. Fig. YCZ-18-treated plants display the BR-deficient phenotype. YCZ-18-treated plants (0.3, 1, 3
μM), Brz220-treated plants (3 μM) and brassinosteroid-deficient mutant (det2) plants were grown
for 6 days in the dark (A) and for 10 days in the light (B-G) on medium containing the chemical
indicated. The control plants (Cont) were untreated. Scale bar = 5 mm.

19. Conti….
• Treated wild-type Arabidopsis plants with YCZ-18 at concentrations
ranging from 0.3 to 3 μM for 5 days after their germination on half
MS agar-solidified medium under light (Fig. 3B-G) and dark (Fig.
3A) condition
• BR-deficient mutant deetiolation2 (det2) and wild-type Arabidopsis
treated with Brz220 (3 μM) were used as positive controls for
comparison with the experimental phenotype.
• The hypocotyl of Arabidopsis seedlings without chemical treatment
elongated to approximately 18 mm with closed cotyledons in the
dark condition (Fig. 3A).
• In contrast, the Arabidopsis wild-type plants in YCZ-18-containing
medium exhibited short hypocotyls and opened cotyledons, similar
to the features of det2 mutant and wild-type plants treated with
Brz220 (Fig. 3A).

20. Fig 4. Effect of YCZ-18 on the hydroponic growth of Arabidopsis. Arabidopsis plants
grown under hydroponic conditions with or without YCZ-18 treatment were treated as
indicated in the methods section. Forty-five-day-old Arabidopsis (A); forty-five-day-old
Arabidopsis treated with YCZ-18 at 0.1 μM (B), 0.5 μM (C), or 1 μM (D). The growth curves
of Arabidopsis treated with different concentrations of YCZ-18 (E). Data are the means ± s.e.
obtained from 8 to 10 plants. Bar = 1 cm.

21. Fig 5. Effect of YCZ-18 on the growth of Arabidopsis in soil. The application of YCZ-18 on wild-
type Arabidopsis was performed by spraying an aqueous solution of YCZ-18 (5 μM) onto ten-day-old
wild-type Arabidopsis plants (approximately 0.2 pmol/plant), as indicated in the methods section. Four-
week-old Arabidopsis seedlings (A), four-week-old Arabidopsis treated with YCZ-18 (B), six-week-old
Arabidopsis (C), six-week-old Arabidopsis seedlings treated with YCZ-18 (D), roosette leaf number of
six-week-old Arabidopsis at bolting from three plants (E). Data are the means ± s.e. obtained from 3
plants. Scale bar = 1 cm.

22. YCZ-18 reduces endogenous BR levels different
from Brz.

23. Fig 6. Longitudinal sections of YCZ-18 treated and untreated plant tissues.
Stem from a seven-week-old control plant grown under hydroponic conditions.
Average cell length of pith is 203.4 ± 12.5 μm (A). Stem from a seven-week-old
YCZ-18-treated (1 μM) plant. Average cell length is 76.0 ± 1.0 μm (B). Arabidopsis
plants were grown as shown in Fig. (A) And (B) are at the same magnification; Bar
= 150 μm.
YCZ-18 induced dwarfism of Arabidopsis owing to a reduction in cell length

24. Research Study

25. FIGURE 1 | Schematic
representation of major abiotic
stresses, their consequences and
the components of plant
antioxidant defense system

26. DROUGHT STRESS
• Spraying with HBL (0.01μM) to 30-day stage seedlings of Brassica juncea
subjected to drought stress (for 7 days at the 8–14 (DS1)/15–21 (DS2) days
stage of growth) improved the activities of antioxidant enzymes such as
CAT, POD and SOD, and the content of proline (Fariduddin et al., 2009a).
• Lycopersicon esculentum, subjected to drought stress and pretreated with
BR showed increased activities of POD, SOD, CAT and APX, and the
contents of non-enzymatic antioxidants such as AsA and proline
(Behnamnia et al., 2009).
• Yuan et al. (2010) also reported that 1.0μM 24-epiBL treatment
significantly alleviated water stress and increased the activities of
antioxidant enzymes such as CAT, APX, and SOD that decresaed the levels
of H2O2 and MDA in two Lycopersicon esculentum genotypes viz., Mill.
cv. Ailsa Craig (AC) and its ABA-deficient mutant notabilis (not).

27. SALINITY STRESS
• Eggplant seedlings, when exposed to 90 mM NaCl with 0,
0.025, 0.05, 0.10, and 0.20 mg dm−3 of epiBL for 10 days
exhibited decreased electrolyte leakage, superoxide
production, MDA, H2O2 probably as a result of increased
activities of SOD, GPX, CAT and APX enzymes and the
contents of non-enzymatic antioxidants such as AsA and
GSH (Ding et al., 2012).
• 24-epiBL decreased the adverse effects of salinity stress on
two varieties of pepper (Capsicum annuum) arguably by
increasing the activities of antioxidative enzymes and the
contents of proline, total anthocyanins and minerals (Abbas
et al., 2013)

28. BRASSINOSTEROIDS SIGNALING

29. PHYSIOLOGICAL ROLE
• GROWTH OF POLLEN TUBES
• PROMOTION OF SEED GERMINATION
• CHILLING INJURY
• PROMOTES RIPENING
• POSTHARVEST DISEASE AND SENESCENCE

30. BRS REQUIRED FOR THE GROWTH OF POLLEN TUBES
• Pollen is a rich source of BRs, and are importance for male fertility.
• BR has been shown to promote the growth of the pollen tube from
the stigma, through the style, to the embryo sac.
Species Tissues Levels (ng g-1 f.w.)
Brassica napus pollen >100
Helianthus annuus pollen 21-106
Vicia faba pollen, seed 5-628
Citrus sinensis pollen 36.2
Zea mays pollen, shoot 2.0-120
Lilium elegans pollen 1.0-50
Table: Distribution and Endogenous Levels of BRs in Selected Plants

31. RESEARCH STUDY

32. PROMOTION OF SEED GERMINATION
• Brassinosteroids (BR) and GA interact with light in regulating
elongation growth of shoots and photomorphogenesis of seedlings
by what appear to be independent pathways (Altmann, 1999).
• Endogenous BRs have been identified in the seeds of several
species, including pea (Yokota et al., 1996).
• BR application has been reported to enhance germination of certain
parasitic angiosperms, cereals, Arabidopsis and tobacco.
• Pretreatment with brassinolide stimulates the germination and
seedling emergence of aged rice seeds and seed treatment of barley
accelerated subsequent seedling growth.
-S.Hayat 2003

33. RESEARCH STUDY…
Figure 1. The accumulation of XET enzyme activity in germinating seeds of Nicotiana tabacum
cv. Havana 245.
Tobacco seeds were imbibed without (Control; C) and with 10 nM brassinolide (BR) in the
medium and incubated for the times indicated (hours) in continuous light; in addition 10 μM ABA
was added to the medium of one series (ABA) (Leubner-Metzger, 2001).
Protein extracts from entire seeds, seed tissues (Endosperm, Embryo), or seedlings were used.
Testa rupture in the populations of ca. 150 seeds was 0 % at 30 h and 100 % at 45 h.
Endosperm rupture was 0 % (45 h), ca. 0 % (Control, 55 h) and ca. 30 % (BR, 55 h); only seeds
without endosperm rupture were used for the extracts.
Tissues were homogenized and XET enzyme activities were determined by the XET 'dot blot'
assay as described by Fry (1997). For the semiquantitative XET assay ca. 3 μl (60 μg protein)
were applied onto the XET 'dot blot' test paper and incubated for 7 h at 25 °C. Elevated
fluorescence under the UV lamp is indicative for accumulating XET enzyme activity.

34. CHILLING INJURY
• The effect of BR on CI of pepper fruit during cold storage is shown in Fig. 1A and B.
In the untreated control pepper fruit, CI symptoms (surface pitting and calyx
discoloration as shown in Fig. 1B) occurred at 3 days after storage (DAS), and the CI
index was as high as 28.12% at 12 DAS and 65.2% at 18 DAS (Fig. 1A).
• In pepper fruit treated with BR (5, 10 and 15 M), CI symptoms occurred at 6 DAS.
Moreover, the CI index in BR-treated fruit was significantly lower compared to the
index in control fruit. Among all the treated fruit, BR at 15 M was the most effective in
alleviating CI of pepper fruit during storage at 3 ◦C. (Qing Wang, et al. 2012)

35. PROMOTES RIPENING
• Brassinosteroids (BRs) are associated with the ripening of berries (Symons et al., 2006), and
exogenous application of 24-epibrassinolide (a BR analog) increases the accumulation of phenolic
compounds (Luan et al., 2013; Xi et al., 2013) such as anthocyanins, which are secondary
metabolites that determine the color of the berries.
• Symons et al. (2006) reported that BRs are involved in the ripening of “Cabernet Sauvignon”
berries.
• Recent studies have shown that BRs are involved in the accumulation of sugar during the ripening of
“Cabernet Sauvignon” (Xu et al., 2015).
• On the other hand, exogenous applications of 24-epibrassinolide during veraison are effective at
increasing sugar accumulations, reducing total acidity at harvest and significantly increasing the total
anthocyanin content in “Cabernet Sauvignon” (Luan et al., 2013; Xi et al., 2013)

36. Conti…

37. Conti…
• Brassinosteroids affect
the accumulation of
phenolic compounds,
including anthocyanins.
• In present study,
compared with the
control treatment,
treatments E-0.4, T-0.4,
T-0.8, and B-2000
effectively increased
CIRG at harvest.
-Alexis E. Vergara et al. 2018

38. Changes of brassinosteroid synthesis
gene expression during tomato fruit
development
• According to recent reports (Jia et al.,
2011; Chai et al., 2013), we define six
ripening stages of “Yuanbao” tomato fruit,
including small green (SG), big green
(BG), mature green (MG), breaking stage
(B), pink stage (P) and red stage (R)
respectively at 7, 14, 24, 29, 32, 35 days
after anthesis (Fig. 1C).
• The transcript levels of brassinosteroids
biosynthetic genes LeDET2 were
increased in SG fruit, but decreased
rapidly up to the B stage, then remain
stable from B to R stages (Fig. 1C).
• This might suggest that feedback
regulation mechanisms of brassinosteroid
signaling are functional during tomato
ripening. This result also suggested that
brassinosteroids might play a role in
tomato fruit ripening.
-Tong Zhu, 2015

39. POSTHARVEST DISEASE AND SENESCENCE
• BR at a concentration of 5 M inhibited decay caused by P. expansum in jujube fruit.
A little less disease incidence (Table 1) and much smaller lesion diameters (Fig. 1)
were observed in BR-treated fruit as compared to control fruit.
• In addition, natural decay in BR treated fruit was much lower than that of control
fruit after 18 d of storage at 25 ◦C (Table 2).
• The results indicated that BR treatment reduced decay in jujube fruit caused by P.
expansum.

40. Conti…
-Zhu Zhu et al. 2020

41. APPLICATIONS OF BRASSINOSTEROIDS
INSECT CONTROL ?
Interfere with ecdysteroids (molting hormones) in insects. The
process of shedding and replacing the rigid exoskeleton is known
as molting.

42. INTERACTIONS OF BR WITH OTHER
PLANT HORMONE
• Recently, a promoter element was identified that is
responsive to both auxin and BR.
• There are also reports that the synergism observed with
BL and GA might be related to the fact that both
hormones increase expression of MERIS, a XET
thought to be involved in loosening of the cell wall.
• A possible interaction of ABA and BL in cell elongation
was seen in experiments with Arabidopsis.
• How these hormones interact to create such a response
remains unknown.

44. APPLICATIONS
• Application to cucumber increase metabolism and removes pesticide
residue.
• 28-homoBL is most effective increase tolerance to high temperature.
• Effective when applied to rice.
• 24-epiBL have protective role in shoot, root length, also on soluble protein,
prolein content and perioxidase.
• Help to overcome unfavourable cultural and environmental condtitons.
• Also use in horticultural crops.
• Helps to bridge the gap between consumers health concerns and the
producers need for growth.
• Does not contribute to co-evolution of pest.
• Also used in tissue cultures, in cell enlargement.
• It was reported the BRs at low conentration acted as inhibitor of growth of
Agrobacterium tumefaciens.

45. CONCLUSION
• Brassinosteroids are a group of steroidal plant hormones occurring
naturally throughout the plant kingdom.
• There are a large number of analogues of BS and the most stable ones
are epibrassinolide, homobrassinolide and brassinolide.
• However, some synthetic and commercial analogues of BS are also
available in the market. BS are reportedly non-toxic and eco-friendly.
• They are extensively exploited in enhancing the productivity of
different agricultural crops.
• They have also been used in enhancing the quantity and quality of some
plants of horticultural importance e.g. tomato, mango, straw berry,
litchi, passiflora, grapes, watermelon etc.
• However, the studies are very less and the field is very vast which has
remained almost untouched. Keeping in view, the great potential of BS,
they can be exploited in vegetables to enhance their production.

46. MORPHACTINS
• Morphactins are the group of substances which act on morphogenesis and
modulate the expression of plants.
• Chemically, they are the derivatives of fluorine compounds.
• Fluorine- inactive, but the addition of COOH group in the 9th position
makes it active.

47. Examples
• Chloroflurenol
• Flurenol
• Methyl benzilate
• Methyl chloroflurenol
• Methyl dichloroflurenol
Chloroflurea

48. EFFECTS OF MORPHACTINS
• Exihibit both synergistic and antagonistic effects depends upon the relative
concentrations.
• They inhibit seed germination, sprouting, growth of seedling and internode
elongation.
• They depolarize cell division which probably leads to distorted morphogenesis.

49. Conti….
• Very effective in inducing lateral bud development, so tillering will be profuse.
• Some morphactins stimulates flowering in certain short day plants.
• Resemble ABA in inducing seed dormancy, bud dormancy and suppressing stem
elongation.
• Most of their effects can be reversed by GA3 treatment.

50. ROLE OF MORPHACTINS
• GROWTH OF SEEDLINGS – INHIBIT
• STEM ELONGATION – DWARFING EFFECT
• FLOWERING PATTERN AND SEX EXPRESSION
• FRUIT AND SEED DEVELOPMENT

51. GROWTH OF SEEDLINGS
• Morphactin inhibited the growth of seedlings (Fig. 1).
At a concentration of 25 mgj l the inhibition of radicle
growth was higher (70%) than that of the hypocotyl
(25%).
• The cotyledons of treated seedlings were of a darker
green colour than those of the control seedlings.
• GA3 (25 mg/1) increased the elongation of hypocotyl
as well as radicle by 75 and 56%, respectively.
• Kinetin (l mg/1) caused 40% increase in the growth of
hypocotyl and radicle and the other cytokinin (BAP, 1
mg/1) caused 50% and 20% enhancement in the
growth of hypocotyl and radicle, respectively.
• GA3 when applied to seeds along with morphactin,
completely reversed the morphactin-induccd
inhibition of seedling growth.
FIG. Effect of different concentrations of
morphactin on growth of the seedlings after
120 hr of germination.
• As mentioned above, morphactin caused 25% inhibition of hypocotyl growth but in the
presence of GAa the hypocotyl showed 66 6% increase in growth.
• Morphactin caused 70% decrease in the growth of radicle but in the presence of GA3 it
showed 20% increase over the control seedlings

53. Conti....
• The two cytokinins also
reversed the morphactin-
induced inhibition of
hypocotyl growth. The
seedlings obtained from
seeds pretreated with
kinetin and
BAP+morphactin showed
33.3% and 41.5% increase
in hypocotyl growth.
Kinetin and BAP could only
reverse 70% morphactin-
induced inhibition of radicle
growth to 20% and 24%,
respectively (Figs. 2 and 3).

54. STEM ELONGATION
a) initial plants on the day of treatment (August 11);
b) control plants, treated with lanolin only; the upper part of the last internode abscised and sprouting of axillary
bud is visible;
c) morphactin; the elongation and thickening of the treated internode can be observed;
d) benzyladenine; the upper part of the last internode abscised and sprouting of axillary bud is visible;
e) morphactin + benzyladenine; the elongation and thickening of the treated internode can be observed.
B: Picture of initial treatment a, and treatment b, c, d and e after removal of all leaves and axillary buds in the
treatments
Fig. 1.
The effect of morphactin IT3456,
benzyladenine and their mixture,
all used at a concentration of
0.2%, applied on the last
internode of decapitated shoots of
Bryophyllum calycinum on the
elongation and thickening of
treated internodes; treatments
made on August 11 (a),
photographed on October 15
A: Picture of treatment a, b, c, d
and e.

55. Fig. 2. The effect of morphactin IT3456 on the histological changes (transections) in cambial
activity, xylem, phloem and cortex formation in the treated internode in the decapitated stem
of Bryophyllum calycinum
a) control; Bar = 150 μm.
b) b) and c) treatment with morphactin (see Fig. 1); Bar = 150 μm.
c) Enlarged cambial zone and cambial derivatives on the phloem and xylem sides Cx,
cortex; CZ, cambial zone; E, epidermis; EZ, expansion zone; MZ, maturation zone and
mature xylem; X, xylem; P, pith; Ph, phloem;

57. Flowering Pattern and Sex Expression

59. FRUIT AND SEED DEVELOPMENT
• This paper reports the effect of a morphactin, chloroflureno-
methylester, on pollen germination, pollen tube growth, and
development of fruit and seed in Nicotiana rustlca.
• (2-chloro-9-hydroxyfluorene-9- carboxylic acid, CFI) at 1,
10, 100, and 1000 ppm, was sprayed with an atomizer on
the inflorescence, and on both surfaces of the leaves.

60. Fig. 1: A-E. Effect of the morphactin CFI on fruit and
seed development in Nicotiana rustica. A
representative fruit from each of the treatments
photographed after removing part of the fruit wall. In
A and B, seeds are normal and compactly arranged.
In C and D, many seeds are degenerated. E lacks
normal seeds, only remnants of degenerated seeds are
visible but the placental enlargement is comparable
to that in the control A x 2· 0, B x 2· 5, C-E x 3·3.

61. Other identified plant growth regulators include:
• Salicylic acid — activates genes in some plants that produce chemicals that aid in the defense
against pathogenic invaders.
• Jasmonates — are produced from fatty acids and seem to promote the production of defense
proteins that are used to fend off invading organisms. They are believed to also have a role in
seed germination, and affect the storage of protein in seeds, and seem to affect root growth.
• Plant peptide hormones — encompasses all small secreted peptides that are involved in cell-
to-cell signaling. These small peptide hormones play crucial roles in plant growth and
development, including defense mechanisms, the control of cell division and expansion, and
pollen self-incompatibility.
• Polyamines — are strongly basic molecules with low molecular weight that have been found
in all organisms studied thus far. They are essential for plant growth and development and
affect the process of mitosis and meiosis.
• Nitric oxide (NO) — serves as signal in hormonal and defense responses (e.g. stomatal
closure, root development, germination, nitrogen fixation, cell death, stress response). NO can
be produced by a yet undefined NO synthase, a special type of nitrite reductase, nitrate
reductase, mitochondrial cytochrome c oxidase or non enzymatic processes and regulate plant
cell organelle functions (e.g. ATP synthesis in chloroplasts and mitochondria).
• Strigolactones - implicated in the inhibition of shoot branching.
• Karrikins - not plant hormones because they are not made by plants, but are a group of plant
growth regulators found in the smoke of burning plant material that have the ability to
stimulate the germination of seeds; Wikipedia.

62. REFERENCES
• Symons, G. M., Davies, C., Shavrukv, Y., Dry, I. B., and Rid, J. B. (2006). Grapes on steroids. Brassinosteroids are
involved in grape berry ripening. Plant Physiol. 140, 150–158. doi: 10.1104/pp.105.070706.
• Luan, L. Y., Zhang, Z. W., Xi, Z. M., Huo, S. S., and Ma, L. N. (2013). Brassinosteroids regulate anthocyanin
biosynthesis in the ripening of grape berries. S. Afr. J. Enol. Vitic. 34, 196–203.
• Xi, Z. M., Zhang, Z. W., Huo, S. S., Luan, L. Y., Gao, X., Maa, L. N., et al. (2013). Regulating the secondary
metabolism in grape berry using exogenous 24-epibrassinolide for enhanced phenolics content and antioxidant
capacity. Food Chem. 141,30563065.doi:10.1016/j.foodchem.2013.05.137.
• Xu, F., Xi, Z. M., Zhang, H., Zhang, C. J., and Zhang, Z. W. (2015). Brassinosteroids are involved in controlling
sugar unloading in Vitis vinifera “Cabernet Sauvignon” berries during véraison. Plant Physiol. Biochem. 94, 197–
208. doi: 10.1016/j.plaphy.2015.06.005 .
• Tong Zhu, Wen-Rong Tan, Xing-Guang Deng, Ting Zheng, Da-Wei Zhang, Hong-Hui Lin. Effects of
brassinosteroids on quality attributes and ethylene synthesis in postharvest tomato fruit. Postharvest Biology and
Technology, 100: 196–204.
• Vergara AE, Díaz K, Carvajal R, Espinoza L, Alcalde JA and Pérez-Donoso AG (2018) Exogenous Applications of
Brassinosteroids Improve Color of Red Table Grape (Vitis vinifera L. Cv. “Redglobe”) Berries. Front. Plant Sci.
9:363. doi: 10.3389/fpls.2018.00363.
• Yamane, H., Abe, H., and Takahashi, N. (1982). Jasmonic acid and methyl jasmonate in pollens and anthers of three
CameN~a species. Plant Cell Phys~ol. 23, 1125-7.
• F. R. Hewitt, T. Hough B, P. O'Neill, J. M. Sasse, E. G. Williams, and K. S. Rowan. Effect of Brassinolide and other
Growth Regulators on the Germination and Growth of Pollen Tubes of Prunus avium using a Multiple Hanging-
drop Assay, Aust. J. Plant Physiol., 1985, 12, 201-11.
• Qing Wang, Tian Dinga, Lipu Gaoa, Jie Pang, Na Yang. Effect of brassinolide on chilling injury of green bell pepper
in storage. Scientia Horticulturae, 144 (2012) 195–200.
• Fujioka S, Takatsuto S, Yoshida S. 2002. An early C-22 oxidation branch in the brassinosteroid biosynthetic
pathway. Plant Physiol. 130:930–39.
• Malíkova, J.; Swaczynova, J.; Kolar, Z.; Strnad, M. Anticancer and antiproliferative activity of natural
brassinosteroids. Phytochemistry 2008, 69, 418–426.

63. THANK YOU