Cultivation & collection by pooja

By: Pooja H. Khanpara
RDGPC , Rajkot

SUBTOPICS

Definition
Characteristics
Classification
Plant Hormones Actions
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Definition

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44
They are various organic compounds other
than nutrients produced by plants that
control or regulate germination, growth,
metabolism, or other physiological activities.
 Also called phytohormone and recently
called growth bioregulators.

4
Plant Hormones & Growth
(abscisic acid)

Definition
Plant hormones, which are active in very low
concentrations, are produced in certain parts of
the plants and are usually transported to other
parts where they bring out specific biochemical,
physiological, or morphological responses.
They are also active in tissues where they are
produced.
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 Each plant hormone evokes many different responses. Also,
the effects of different hormones overlap and may be
stimulatory or inhibitory.
 The commonly recognized classes of plant hormones are the
auxins, gibberellins, cytokinins, abscisic acid, and ethylene.
 Some evidence suggests that flower initiation is controlled by
hypothetical hormones called florigens, but these substances
remain to be identified.
 A number of natural or synthetic substances such as brassin,
morphactin, and other growth regulators not considered to be
hormones but influence plant growth and development.
6

Definition
Plant hormones (or plant growth regulators, or PGRs)
are internally secreted chemicals in plants that are
used for regulating the plants' growth.
According to a standard definition, plant hormones
are:
Signal molecules produced at specific locations, that
occur in very low concentrations, and cause altered
processes in target cells at otherlocations.
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Characteristics

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  The concentration of hormones required for the
plant response is very low(10-6 to 10-5M),
comparing with the requirement of mineral and
vitamin for plants.
 The synthesis of plant hormones is more diffuse
and not always localized.


Classes of Plant Hormones :
 It is accepted that there are two major classes of plant
hormones:
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Class Action Examples
Promoters- Cause faster
growth
Auxins
Cytokinins(cks)
Gibberellins (gas)
Brassinosteroids
Inhibitors- Reduce growth Ethylene
Abscisic acid (ABA)
Jasmonic acid


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What do hormones control in plants?
  Roots and shoots growth
  Seed germination
  Leaf fall
  Disease resistance
 Fruit formation and ripening
 Flowering time
 Bud formation
 Anything related to plant growth!

Auxins

tryptophan
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Auxins

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 Introduction:
 Auxin is a general name for a group of hormones
that are involved with growth responses (i.e.,
elongate cells, stimulate cell division in callus).
  Not surprisingly, the term "auxin" is derived from
the Greek word "to increase or grow".
 This was the first group of plant hormones
discovered.

Discovery
of Auxins:
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Auxins

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Site
Auxin is made in actively growing tissue
which includes young leaves, fruits, and
especially the shoot apex.
Made in cytosol of cells .

Auxins

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Transport :
 Basipetal (or Polar) Transport Auxin is
transported in a basipetal (towards the base,
base-seeking) direction.
 In other words, auxin moves from the shoot
tip towards the roots and from the root tip
towards the shoot.

1. Cellular Elongation:
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 Auxin can induce and amplify proton pumping.
 Acidified cell walls have increased elasticity which
lead to cell elongation.

Auxin Actions

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2. Cell differentiation
Auxin promotes differentiation of vascular tissue (i.e.,
xylem & phloem):
Auxin and sugar -----> Vascular tissue
Auxin and low sugar (1.5 - 2.5%) -----> Xylem
Auxin and high sugar (4%) ------->- Phloem
Auxin and moderate levels of sugar (2.5 - 3.0%) ----->-
Xylem & Phloem

3. Ethylene production
IAA apparently stimulates the production of
ethylene.
4. Inhibition of root growth
 [IAA] > 10-6 M inhibit root elongation.
 However, very low [IAA] (>10-8 M) favor
root elongation.
5. Stimulate root initiation (lateral roots,
adventitious roots)
Roots always form at the basal end of cutting
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Formation of
adventitious roots
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Auxin Actions

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6. Flowering
Although most plants don’t initiate the
production of flowers after auxin treatment,
pineapple and its relatives (Bromeliaceae) do.
 Once flowers are initiated, in many species,
IAA promotes the formation of female flowers.


7. Parthenocarpic(seed less) fruit development
 Pollination of the flowers of angiosperms initiates
the formation of seeds.
 As the seeds mature, they release auxin to the
surrounding flower parts, which develop into the
fruit that covers the seeds.
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Auxin Actions

Auxin produced by seeds promotes ovary
tissue growth
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8 Apical Dominance
 Lateral branch growth are inhibited near the shoot
apex, but less so farther from the tip.
 Apical dominance is disrupted in some plants by
removing the shoot tip, causing the plant to become
bushy.
Auxin Actions
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
Plant b has apical bud removed so
axillary buds grow
Auxin Actions
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8- Apical Dominance


10- Tropic responses
Such as gravitropism and phototropism
A-Phototropism
 is a growth movement induced by a light
stimulus
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Auxin Actions


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Phototropism
 Sunlight breaks down auxin
 Plant stems indirect sunlight will have the least
amount of auxin
 Area of the plant that is more shaded will have
more auxin
 More cell growth on shaded side
 Plant bends towards light
Auxin Actions


 Light directly over the
plant
 Auxins are in equal
quantity
 Cell elongation is equal
on all sides of the cell
 Greater light on the right
side of the plant
 Auxin quantity becomes
greater on the left cell
 Auxins trigger cell
elongation on the left side
 Plant ‘stretches’ to the
Phototropism
Auxin Actions
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Auxin Actions
 Geotropism or Gravitropism
  The plant stem that was once upright is on its side
  The auxin are settle on the bottom side of the stem
  More auxin accumulate on the stems bottom side
  More cell growth occurs on bottom side
  Plant bends upward
  A growth response to gravity which causes roots to
grow downward and shoots to grow upward
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
Gravitropism
Auxin Actions
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Gibberellins

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  Gibberellins are plant hormones that promote
 growth, seed germination and leaf expansion.
 They occur at low concentrations in vegetative
tissues but at higher concentrations in germinating
seeds.
 Induce cell elongation and cell division.
 Important for plant growth and development
 through flowering and/or seed germination.

Site : Young leaves, roots, and developing seeds
(developing endosperm) and fruits.
 Transport :
  Made in the tissue in which it is used
  Transport occurs through xylem, phloem, or
cell-to-cell.
  Phloem seems to be most important transport
route
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  When applied to intact plants, GA usually causes
an increase, unlike auxin.
  It overcomes dwarfism (small) in mutants
that have a mutation in the GA synthesis
pathway.
 dwarf = short; wild
type = tall ;
 dwarf + GA = tall.
  Thus, GA application:
 (1) stimulates elongation; and
(2) acts on on whole plants.
Gibberellins Actions
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1- Promotes stem elongation

1- Promotes stem elongation
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
2- Overcomes dormancy in seeds
 Gibberellins also have a fundamental role in breaking seed
dormancy and stimulating germination.
 The endosperm of many seeds contains protein and
carbohydrate reserves upon which a developing embryo
relies for energy and nutrition.
 These reserves must be mobilised and transported to the
embryo.
 A range of hydrolytic and proteolytic enzymes break down
endosperm starches and proteins into smaller, more easily
transported molecules, such as sugars and amino acids.
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2- Overcomes dormancy in seeds
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
3- Involved in parthenocarpic fruit development
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External
application of
gibberellins can
also enlarge fruit
size in grapes

4- GA can induce fruit enlargement
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
5- Promotes cell
division & elongation
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6- Flowering
 GA stimulates bolting in Long Day plants and
can substitute for long days or cold treatments
that are necessary for flowering.
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
7-Sex expression
In plants with separate male and female
flowers, GA application can determine sex.
 For example, in cucumber and spinach, GA
treatment increases the proportion of male
flowers.
 In maize, GA treatment causes female flower
development.
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Cytokinins

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Cytokinins are hormones that stimulate cell
division, or cytokinesis
These hormones may also be involved in
controlling leaf senescence and the growth of
lateral branches
The most active, naturally-occurring cytokinin is
zeatin.
Cytokinins occur in most plants including mosses,
ferns, conifers, algae and diatoms


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Site:
Synthesized primarily in the meristematic region
of the roots.
This is known in part because roots can be cultured
(grown in Artificial medium in a flask) without added
cytokinin, but stem cells cannot.
 Cytokinins are also produced in developing embryos
.

Cytokinins

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Transport:
 Via xylem (transpiration stream).
 Zeatin ribosides are the main transport form; converted
to the free base or glucosides in the leaves.
 Some cytokinin also moves in the phloem.

1- Control morphogenesis

In plant tissue cultures, cytokinin is required for the
growth of a callus (an undifferentiated, tumor-like mass of
cells):
Cytokinins Actions
Ratio of cytokinin and auxin are important in determining
the fate of the callus:
The Medium The callus differentiation
callus + auxin + no cytokinin little growth of callus
callus + auxin + cytokinin callus grows well, undifferentiated
The concentration The callus differentiation
callus + low [cytokinin/auxin] callus grows well, forms roots
callus + high [cytokinin/auxin] callus grows well, forms meristem & shoots

Cytokinins Actions
1- Control morphogenesis
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Cytokinins Actions

2- Regulates the cell cycle/cell division
 (hence, the name "cytokinins) –especially by
controlling the transition from G2 mitosis.
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3- Bud development
Direct application of cytokinin promotes the
growth of axillary buds
Exogenous cytokinin and auxin are thus
antagonistic in their effects on axillary bud growth
Cytokinins Actions
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4- Delay senescence
 Senescence is the
programmed aging process
that occurs in plants.
 Loss of chlorophyll, RNA,
protein and lipids.
 Cytokinin application to an
intact leaf markedly reduces
the extent and rate of
chlorophyll and protein
degradation and leaf drop.
Cytokinins Actions
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Cytokinins Actions

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5- Greening
Cytokinins promotes the light-induced formation of
chlorophyll and conversion of etioplasts to
chloroplasts (greening process).

Cytokinins Actions
6. Promote cell expansion
 Cytokinins stimulate the
expansion of cotyledons.
The mechanism is
associated with increased
plasticity of the cell wall, not
associated with acidification.
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
Inhibits growth
Promotes dormancy
Closes stomata
Produced in response to stress.
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Abscisic acid

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 Sites :
 Plastids
  Most tissues, especially leaves and seeds
 Transport :
  Xylem and phloem (greater amounts)


1- ABA—drought resistance
 Abscisic acid is the key internal signal that facilitates
drought resistance in plants
 Under water stress conditions, ABA accumulates in
leaves and causes stomata to close rapidly,
reducing transpiration and preventing further
water loss.
 ABA causes the opening of efflux K+ channels in
guard cell plasma membranes, leading to a huge loss
of this ion from the cytoplasm.
 The simultaneous osmotic loss of water leads to a
decrease in guard cell turgor, with consequent closureof stomata.
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
2- ABA—freezing resistance
 Elevated ABA levels are associated with increased
freezing resistance.
 ABA appears to mediate a plant’s response to
environmental stresses, such as freezing, by
regulating gene expression.
 Certain genes are switched on by ABA while
others are switched off.
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Fig. 17.15: Celmisia aseriifolia (snow daisy)3/2/2014 62

Ethylene

H

H
/
C = C
/
H

H
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
 Ethylene is the only gaseous plant hormone (C2H4)
 It is produced naturally by higher plants and is able to
diffuse readily, via intercellular spaces, throughout the
entire plant body
 Ethylene is involved primarily in plant responses to
environmental stresses such as flooding and drought,
and in response to infection, wounding and mechanical
pressure
 It also influences a wide range of developmental
processes, including shoot elongation, flowering, seed
germination, fruit ripening and leaf abscission and
senescence
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Ethylene Action
1- Ethylene—signal transduction

 Several transmembrane
proteins have been
identified that bind to
ethylene at the cell
surface and function as
signal transducers.
17-65


2- Ethylene—fruit ripening
melons, kiwi fruit and bananas3/2/2014 66
 Under natural conditions, fruits undergo a series of
changes, including changes in colour, declines in organic
acid content and increases in sugar content
 In many fruits, these metabolic processes often coincide
with a period of increased respiration, the respiratory
climacteric
 During the climacteric there is also a dramatic increase in
ethylene production
 Ethylene can initiate the climacteric in a number of fruits
and is used commercially to ripen tomatoes, avocados,

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Ethylene Action
2- Ethylene—fruit ripening

Ethylene Action
3- Ethylene—Shoot Growth
 Applied ethylene has the capacity to influence shoot
growth
 Application of ethylene to dark-grown seedlings can
cause reduced elongation of the stem, bending of the stem and
swelling of the epicotyl or hypocotyl.
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10
4


4- Ethylene—flowering
 The ability of ethylene to affect flowering in
pineapples has important commercial
applications.
 Ethylene also promotes flower senescence
(ageing) in plants such as petunias, carnations
and peas.
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10
5

4- Ethylene—flowering
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10
6


Fig. 17.19: Senescence in carnations
(a)
3/2/2014
10
7
(b)

 5- Thigmomorphogenesis (touch)
  The change in growth form in response to a
mechanical stimulation such as touch.
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10
8


 Brassinosteroids (BRs) are plant steroid
hormones that have a similar structure to
animal steroid hormones
 They have multiple developmental effects on
plants, including :
promotion of cell elongation, cell division
and xylem differentiation, and delaying of
leaf abscission
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10
9

Brassinosteroids

17-74
 BR-deficient mutants exhibit dramatic
growth defects, including dwarfism, reduced
apical dominance and male fertility, as well
as delayed senescence and flowering
 Brassinosteroids switch on specific genes by
inactivating a protein that otherwise
indirectly blocks transcription of those genes


Fig. 17.20: Signal transduction chain for the response to
brassinosteroids3/2/2014 75

POLYPLOIDS
PLOIDY - the number of sets of chromosomes in cell, or in the
cells of an organism
POLPLOIDY - more than two sets of chromosomes are present
polyploidy
autopolyploid
Addition of one
more extra sets of
chr. From same spp.
allopolyploidy
Combination of
chr. sets b/w
different spp.

 Conservation is the process of management
of biosphere in order to obtain the greatest
benefit for the present generation and
maintaining the potential for future.
 Conservation of plant resources is of global
concern because we don't know what we are
losing and what we will need in future.

 To meet the requirements of expanding regional and international
markets healthcare products and needs of growing populations,
large quantities of medicinal plants are harvested from forests
(Desilva,1997).
 In India large number of medicinal plants are extracted from the
wild to meet the increasing demand for raw material needed for
domestic consumption and for export. As a result, the natural
sources are rapidly depleting.
 Medicinal plants contribute to health, income, agro forestry
system, cultural identity and livelihood security.
 Hence there is a need for conservation, cultivation, maintenance
and assessment of germplasm for future use.

 Conservation of biological diversity involves protecting,
restoration and enhancing the variety of life in an area so
that the abundance and distribution of species and
communities contributes to sustainable development.
 The ultimate goal of conservation biology is to maintain
the evolutionary potential of species by maintaining
natural levels of diversity which is essential for species
and populations to respond to long and short term
environmental changes in order to overcome stochastic
factors failing which would result in extinction.

 The two main strategies are ex situ (protection of species
outside their natural habitats) and in situ (in their
natural surroundings) conservation.
 There is a need for coordinated conservation efforts
based on these strategies.
 More information is required on medicinal plant
production, utilization, trade, monitoring the stock of
medicinal plants, development of sustainable harvesting
practices, preservation of traditional knowledge and
intellectual property rights.

 On going efforts in India include both in situ and ex situ
conservation measures viz, plant tissue culture,
introduction of new crop genetic resources, research in
habitat restoration, pollution abatement, seed storage and
tissue banking etc. (Jackson and Sutherland, 2000).

 In situ or on site conservation involves maintaining genetic
resources in their natural habitats i.e., within the ecosystem to
which it is adapted, whether as wild or crop cultivar in farmer's
field as components of the traditional agricultural systems
(Damania, 1996).
 The key operational steps for establishing in situ gene banks for
conservation of prioritized medicinal plants include: Threat
assessment, establishment of a network of medicinal plant forest
reserves, involving local stakeholders, botanical, ecological, trade
and ethno-medical surveys, assessing intraspecific variability of
prioritized species, designing species recovery programmes,
establishment of a medicinal plant seed center etc.
 Conclusively, no in situ conservation project can succeed
without the complete cooperation and involvement of local
people (Srinivasamurthy and Ghate, 2002).

 Ex situ conservation, involves conservation of biodiversity outside
the native or natural habitat where the genetic variation is
maintained away from its original location.
 The ex situ genetic conservation fulfills the requirement of present
or future economic, social and environmental needs. Conservation
also includes propagation and assessment of molecular diversity
(Olorode, 2004)
 Conservation of medicinal plants include a combination of methods,
depending on factors such as geographic sites, biological
characteristics of plants, available infrastructure, and network
having an access to different geographical areas, human resources
and number of accessions in a given collection (Rajasekharan and
Ganeshan, 2002).

 In vitro regeneration include plant/explant growth,
maintenance under disease free condition, retention of
regenerative potential, genetic stability, and ensuring that
there is no damage to the live material.
 It offers a number of advantages over the in vivo method:
 a) great savings in storage space and time
 b) possibility of maintaining species for which seed
preservation is impossible or unsuitable and
 c) disease-free transport and exchange of germplasm, since
cultures are maintained under phytosanitary conditions
(Natesh, 2000)

 In vitro multiplication protocols for fast propagation of a number
of red listed medicinal, aromatic and recalcitrant taxa that are
difficult to propagate through conventional means would be very
useful.
 Usually, shoot tips or axillary buds are cultured on a nutrient
medium containing (i) high levels of cytokinins or (ii) low
concentrations of auxin coupled with high-cytokinin content.
Somatic embryos, or even axillary buds are encapsulated in
hydrosoluble gels to form 'artificial seeds' and have used for rapid
propagation of the species.
 Even more important is the reintroduction of in vitro raised
material into their natural habitat and monitoring its
performance over several years, to ensure fidelity with respect to
active compounds or the marker chemical, vis-a-vis the parents
(Natesh, 2000).

 The cell culture process itself can result in genetic changes in the
regenerated plants. These heritable genetic changes are termed as
somaclonal variation.
 The presence of an undifferentiated callus phase in the regeneration
protocol enhances the chances for somaclonal variation among the
regenerated plants.
 These variations can result from simple DNA sequence differences.
The cell environment appears to induce a very high frequency of such
mutations.
 Other types of changes that frequently occur in regenerated plants
could be due to chromosomal, structural and number changes due to
rearrangements in multi-gene families, gene silencing due to changes
in DNA methylation, action of jumping genes etc.
 Hence, it is necessary to avoid the use of auxin and auxin like
substances in the meristem multiplication protocols.
 It is also mandatory to check the fidelity of the plants multiplied from
the meristem cultures and plants multiplied from cryo preserved

 Cryopreservation of plant cells and meristems is an important tool
for longterm storage of germplasm or experimental material without
genetic alteration using a minimum space and maintenance.
 The development of methods to store apical meristems in liquid
nitrogen successfully is needed to aid in the conservation of genetic
resources.
 Cryobanks are basically meant for storage of germplasm. For
longterm preservation, cryogenic storage at ultra low temperatures
under liquid nitrogen (-150 to -196°C) is the method of choice.
Relatively new to plants, cryopreservation has followed advances
made in the mammalian systems is achieved either through slow
cooling or vitrification.
 Encapsulation/dehydration is another new technique that offers
practical advantages.

 It is based on the technology originally developed for
production of synthetic seeds, i.e., somatic embryos
encapsulated in a hydrosoluble gel.
 Several types of in-vitro raised materials such as
meristems/shoot tips, cell suspensions, protoplasts, somatic
embryos and pollen embryos of medicinal and aromatic
species have been studied from the cryopreservation
perspective (Natesh 2000).

 Preservation by under-cooling has recently been applied to plant
tissue cultures.
 The objective of this approach is to maintain tissues at low
temperatures (-10 to -20 °C) but in the absence of ice crystallization.
The plant tissues are immersed in immiscible oil and the emulsion
thus formed can be under cooled to relatively low temperatures
thereby circumventing ice formation, one of the most injurious
consequences of low temperature storage.
 Although good recovery has been reported in certain species, this 9
has only been achieved using a temperature of -10° C and for
relatively short storage periods (6-48 hours).

 Recently, vitrification, simplified freezing, and encapsulation-
dehydration methods have been used for storage of valuable
germplasm.
 These new procedures may replace freeze-induced cell dehydration by
removal of all or of a major part of freezable water from cells at room
temperature or at 0° C.
 In the encapsulationdehydration technique, extraction of water results
in progressive osmotic dehydration, additional loss of water is
obtained by evaporation and the subsequent increase of sucrose
concentration in the beads.
 In the technique, preculturing encapsulated meristems in medium
enriched with sucrose before dehydration induces resistance to
dehydration and deep-freezing.
 The vitrification procedure for cryopreserving meristems involves
preculture and/or loading and osmotic dehydration by short exposure
of meristems to highly concentrated mixture of cryoprotectants.
 The encapsulation-dehydration technique is easy to handle and

 Usually seeds, being natural perennating structures of plants,
represent a condition of suspended animation of embryos, and are best
suited for storage.
 By suitably altering their moisture content (5-8%), they can be
maintained for relatively long periods at low temperatures (-18 °C or
lower).
 However, in several species, rhizome/bulb or some other vegetative
part may be the site of storage of active ingredients, and often, such
species do not set seed.
 If seeds set, they may be sterile or recalcitrant i.e., intolerant of
reduction in moisture or temperature, or, otherwise unsuitable for
storage.
 It is now possible to store materials other than seed, such as pollen or
clones obtained from elite genotypes/cell lines with special attributes,
in-vitro raised tissues/organs, or, genetically transformed material
(Natesh 2004).

 The IUCN Red Data book lists 34,000 plants with endangered
status.
 The Botanical Garden Conservation International (BGCI) 2000
database indicates that there are about 1846 botanic gardens. In-
order to put efforts for ex-situ conservation; these botanical gardens
have to cultivate several hundreds of endangered, rare and
vulnerable plant species, which requires elaborate facilities and
extraordinary efforts.
 Therefore, biologists feel that the ex situ conservation should be
considered as a complimentary measure of in situ conservation for
holistic strengthening of conservation.

Cultivation & collection by pooja

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