Techniques of in vitro clonal propagation for fruit crops

2. Techniques of In vitro clonal
propagation for fruit crops
Pawan Kumar Nagar
M.Sc. (Horti.) Fruit science
REG. NO: 04-2690-2015
PRESENTED BY

3. In vitro:
The culture of plant cells or organs in culture vessels (like
test tubes) under controlled environment and nutritive growth medium.
In vitro clonal propagation (Micropropagation):
Micropropagation is the production of new plants under the ultra-
controlled environment within the culture vessel (in vitro). Commercial
micro-propagation began in the United States in 1965 with orchid
production.
Uses for micropropagation:
 Mass propagation of specific clones
 Production of pathogen-free plant
 Clonal propagation of parental stock (inbred lines) for hybrid seed
production
 Year-round nursery production
 Germplasm preservation.
Techniques of In vitro clonal propagation for fruitcrops

4. Commercial Tissue culture propagation is particularly
useful in the following situations:
 Where the propagation rate is slow by conventional means
 New cultivars with high market demand
 Cultivars with high market value
 Plants which is difficult to propagate clonally
 Conservation of endangered species.

5. Disadvantages of Tissue culture propagation:
 High labor costs
 A requirement for expensive and sophisticated facilities, trained
personnel, and specialized techniques
 A high-volume, more or less continuous distribution system, or
adequate storage facilities to stockpile products, is required
 Pathogen contamination or insect infestation can cause high
losses in a short time
 Variability and production of off-type individuals can be a risk
in the products emerging from micropropagation
 Careful rouging field testing of new products, and continuing
research and development are essential to decrease this risk
 Economics and marketing are key to the success of commercial
operations

6. General laboratoryfacilities andprocedures:
Facilitiesand Equipment:
Facilities for micropropagation may be placed into three
categories as determined by their scope, size, sophistication, and
cost. These categories are:
 Research laboratories where precise work requires highly
sophisticated equipment
 Large commercial propagation facilities where several
million plants can be mass-produced annually and
 limited facilities for small research laboratories, individual
nurseries, or hobbyists where a relatively small volume of
material is handled.
Regardless of size, the facility should include separate
preparation) transfer, and growing areas. In addition, there may
be a need for service areas, office, and cold storage.

7. MediaPreparation:
Success of in vitro technology depends on the nutritional
media. It is essential to understand the nutritional requirement for
optimal growth of a particular tissue. Medium ingredients can be
grouped into the following categories:
 Inorganic salts
Organic compounds
Gelling agents
There are basically four stages to the micropropagation process.
Stage I. Establishment
Stage II. Shoot multiplication
Stage III. Root formation
Stage IV. Acclimatization
Micropropagation procedures:

8. Stage I-Establishment:
The function of this stage is to disinfest the explant, establish it in
culture, and then stabilize the explants for multiple shoot development.
Preparation for Establishing Cultures:
 Handling Stock Plants:
A principal consideration for handling stock plants is reducing the
potential for contamination by fungi, viruses and other pathogens.
Pathogens are not automatically eliminated by in vitro technique unless
this goal is built into the system. Selected “virus-free” or pathogen-indexed
stock plants should be used.
 Choice of Explant
The kinds of explant and where and how they are collected varies
with the purpose of the culture, the species, and often the cultivar.
 Disinfestation:
Disinfestation is the process of removing contaminants from the
surface of the explant. A typical procedure would be to cut the plant
material into small pieces and wash them in running tap water. For
materials that are difficult to disinfest, a quick dip in alcohol may be
helpful. Wrapping the shoots with small squares of sterile gauze will hold
them intact during the treatment.

9. Pretreatment of Explants:
Exudation of phenolic and other substances from the cut explant
surface can inhibit initial shoot development. Using liquid media in the
initial stage or frequent transfers on agar medium for several days may leach
out the toxic materials. Likewise, using antioxidants, such as ascorbic acid
and citric acid, in the preliminary washing may be useful.
Initial Shoot Development:
Depending on the explant, shoots will initiate from:
(a) The stimulation of axillary shoots
(b) The initiation of adventitious shoots on excised shoots, leaves, bulb,
scales, flower stems, cotyledons, and other organs
(c) The initiation from callus on the cut surfaces. Which medium is selected
varies with the species, cultivar, and kind of explant to be used
Some woody plant species can take up to a year to complete Stage I, this
phenomenon is called stabilization. A culture is stabilized when explants
produce a consistent number of “normal” shoots after subculturing.
“Abnormal” shoots usually are arrested in development and fail to elongate.

10. Establishment stage

11. Stage II-Shoot Multiplication:
In the multiplication stage each explant has expanded into a
cluster of microshoots arising from the leaf axils or base of the
explant. The separate microshoots are transplanted into a new culture
medium, which is a process called subculturing. During the
multiplication stage, cultures are subcultured every 4 to 8 weeks.
The kind of medium used depends on the species, cultivar,
and type of culture. The basal medium is usually the same as in Stage
I, but often the cytokinin and mineral supplement level is increased.
Adjustments may need to be made after some experimentation.

12. Stage III- Root Formation:
Shoots developed during the multiplication stage do not usually
have roots. There are some exceptions (like African violet and poplar)
that spontaneously root on the multiplication medium, but for most other
species, single shoots (micro-cuttings) must be moved to a medium or
suitable environment to induce roots. Therefore, the purpose of Stage III
is to prepare plantlets for transplanting from the tissue culture
environment of the test tube to a free-living existence in the greenhouse
and on to their ultimate location. Therefore, Stage III may not only
involve rooting, but also conditioning the plantlet to increase its potential
for acclimation and survival during transplanting, for example, by
increasing agar and or sucrose concentration. Light intensity is
sometimes increased during Stage III. Some plants respond to an
“elongation” phase between Stages II and III, achieved by placing the
microshoot into an agar medium for 2 to 4 weeks without cytokinins (or
at very low levels) and, in some cases gibberellic acid, which reduces the
influence of cytokinin. The microshoots are then rooted in a cytokinin
and GA-free medium.

13. Root formation stage

14. Stage IV- Acclimatization to Greenhouse Conditions:
Once plantlets are well rooted, they must be acclimatized to the
normal greenhouse environment. In vitro rooted plants are removed from
the culture vessel and the agar is washed away completely to remove a
potential source of contamination. Plantlets are transplanted into a
standard pasteurized in a shaded, high humidity tent or under mist or fog.
Several days may be required for new functional roots to form.
Plantlets should be gradually exposed to a lower relative humidity and
higher light irradiance. Any dormancy or resting condition that develops
may need to be overcome as part of the establishment process. In
summary, micropropagation involves the vegetative propagation of
plants through (1) establishment, (2) multiplication, (3) rooting, and (4)
acclimatization (transplanting) of clones in vitro starting with small
explants and ending with a rooted plant established in a container or in
the ground.

15. Acclimatization

16. Types of tissue culture systems
The procedures used for tissue culture that are associated
with propagation. These include the formation of:
1. Plantlets
2. Seedlings
3. Callus
4. Somatic embryos
Of these techniques, the process of plantlet formation via
micropropagation has the most direct application for plant
propagation and will be emphasized in this chapter.

17. Micropropagation of Plantlets from Tissue Culture:
Cultural systems described under plantlet formation are based
on the maintenance and multiplication of microshoots in culture to
produce rooted micro cuttings. A distinction is made between
microshoots originating from axillary buds and those arising
adventitiously directly from tissue without axillary buds (i.e., leaf or
petiole) or indirectly from callus developed from the original explant.
Developmental Stages in Micropropagation:
Success of micropropagation is largely due to separating
different developmental aspects of culture into stages, each of which is
manipulated by media modification and environmental control. Four
distinct stages are recognized for most plants.
Stage I: Establishment
Stage II: Shoot multiplication
Stage III: Root formation
Stage IV: Acclimatization

18. There are basically two developmental patterns for plantlet
formation in tissue culture, described by the way shoots originate from the
initial explant in Stages I and II. Once shoots have formed, the rooting and
acclimatization stages are the same for the various developmental patterns.
Meristem culture:
 A procedure to eliminate diseases from plants, using a very small piece
of tissue from the shoot tip as the initial explant
 Meristem culture utilizes the smallest part of the shoot tip as the
explant, including the meristem dome and a few subtending leaf
primordia
 The number of additional structures depends on the length of the
excised stem. The primary reason for this procedure is to produce a
plantlet that is free of systemic viruses, virus-like organisms, and
superficial fungi and bacteria
 The meristem is usually free of disease organisms; therefore, the
smaller the explant, the more effective the elimination of pathogens
Invitro techniques of clonal propagation(Tissue culture):

20. Shoot-tipmicrografting in vitro
Grafting very small meristem tips comparable to those
described in the preceding section can be used as an alternative
method to produce virus-free materials for various woody plants, such
as Citrus and Prunus. For example, this procedure is important in
citrus not only because it is successful, but also because explants can
be used from ontogenetically mature trees, which avoids the juvenile
phenotype of nucellar seedlings also used in virus cleanup in citrus.
 The technique for micrografting can be illustrated with citrus.
Citrus embryos are excised from rootstock seeds, surface
disinfested, and planted in standard inorganic salt medium with 1
per cent agar
 Embryos germinate in the dark in 2 weeks
 Seedlings are then removed and decapitated to a 1 to 1.5 cm
length; cotyledons and lateral buds are excised with a mounted
razor blade

21.  A 0.14 to 0.18 mm tip with 3 leaf primordia is used as the scion
and this gives reasonable success, and the shoot tip eliminates
viruses
 An inverted T-bud cut is made in the seedling root stock, cutting
1 mm down the stem, followed by a horizontal cut on the
bottom
 The excised shoot tip is placed inside the flap next to the
cambium
 Grafted plants are placed in a liquid medium
 A filter paper bridge with a center hole supports the stem
 Cultures are kept in the light for 3 to 5 weeks to heal. When two
expanded leaves appear on the scion, the grafted plant is
transplanted
 A similar procedure has been used for apple and plum
 Rootstocks are either seedling plants or rooted stems
 Shoot-tip scions taken from cultured plants reduce
contamination problems

22. Shoot tip micrografting in vitro

23. Anther culture:
 Anther culture is a procedure for obtaining haploid plants
from normally diploid plants
 A flower bud from Nicotiana tabacum is excised just as the
petals are emerging from the bud
 The immature anther is removed aseptically and planted on
agar with a standard nutrient medium without hormones
 Pollen should be at the uninucleate, microspore stage of
development
 Somatic embryos develop from callus derived from the
haploid microspores
 Haploid (1n) embryos germinate to form plantlets
 It is used for plant breeding to produce haploid plants

24. Guha and Maheshwari (1964)
Anther culture

25. EmbryoCulture and EmbryoRescue:
 Embryo culture is a procedure involving tissue culture of
immature embryos that require controlled conditions to
complete development
 It is most commonly used by plant breeders for embryo rescue
of genetic crosses that would not form seed on the plant
 Embryo culture involves the excision of an embryo from a
seed and germinating it in aseptic culture
 As early as 1904, the first attempts were made to grow
immature embryos in nutrient solution
 These immature crucifer embryos germinated, but growth was
weak. Hannig called this “precocious germination.” A principal
use of embryo culture is to “rescue” embryos that would have
aborted within the seed before the fruit was mature
 Much interspecific and intergeneric hybridization are initially
successful but the embryo aborts during development.

26. Embryo rescue or embryo culture

27. Ovary and Ovule Culture:
 Ovary and ovule culture include the aseptic culture of the excised
ovary, ovule (fertilized or unfertilized), and placenta attached
within the ovule
 Although this technique was first utilized to investigate problems
of fruit and seed development, adaptations of the procedure have
uses in propagation, particularly in genetic improvement
 Unfertilized ovules have been excised, grown in culture, supplied
with pollen, and subsequently fertilized in vitro
 Such procedures are most successful with plants that have multiple
ovules
 Pollen can be placed directly on the placenta inside the ovule,
where pollen tubes can develop and grow immediately into the
ovules without passing down the style
 Cultured ovules are useful for rescuing embryos that abort at a very
young stage if not separated from the plant
 This technique is simpler than culturing isolated embryos.

28. Ovary and ovule culture

29. Callus Culture:
 Callus results from cell division in non-differentiated parenchyma
cells
 Eventually a callus culture does form stratified cell layers with outer
meristematic layers and inner cells that can form vascular tissue.
Callus is produced on explants in vitro as a response to wounding
and growth substances, either within the tissue or supplied in the
medium
 Explants from almost any plant structure or part seeds, stems, roots,
leaves, storage organs, or fruits can be excised, disinfested, and
induced to form callus
 Continued subculture at three to four week intervals of small cell
clusters taken from these callus masses can maintain the callus
culture for long periods

30. Callus Formation

31. Cell Suspensions culture:
 A suspension culture is started by placing a piece of friable callus or
homogenized tissue in liquid medium so that the cells disassociate from each
other
 Batch cultures, cells are grown in a flask placed on a shaking device that allows
air and liquid to mix. Rotating devices that result in continuous bathing of the
tissue are available
 Another device, called a chemostat or turbidostat, continuously cycles the media
through the cell culture essentially in the same manner as in the culture of
microorganisms
 In a third method, cells on a filter paper layer are placed on a shallow liquid
medium in a Petri dish with no agitation
 Growth of cells follows a typical pattern based on changes in rates of cell
division
 Cells first divide slowly (lag phase), then more rapidly (exponential), increasing
to a steady state (linear), followed by a declining rate (deceleration) until a
stationary state is reached
 When cells are transferred to a new liquid medium, the process will be repeated.
Under proper environmental conditions with media control, the process can go
on indefinitely.

32. Cell suspension culture

33. Protoplast Culture:
 Protoplasts are the living parts of plant cells, containing the
nucleus, cytoplasm, vacuole and various cellular structures
surrounded by a semipermeable membrane (plasmalemma)
but with the cell wall removed protoplasts can be obtained
from cells in suspension or derived directly from mesophyll
leaf cells
 The major advance that permitted protoplast cultures to be
made was the discovery that plant cell walls could be
removed by enzymes that digest pectin and allow the
protoplast surrounded by its cellular membrane to survive.

37. 1.Banana:
 Method: Shoot-tipcultures:
Banana is commercially propagated by shoot tip culture in vitro.
 StageI: Initiationof shoot cultures:
Shoot cultures of banana start conventionally from any plant part
that contains a shoot meristem, i.e. the parental pseudo-stem, small
suckers, peepers and lateral buds. The apex of the inflorescence and
axillary flower buds are also suitable explants for tissue culture initiation.
Overall, it is important to select explant material from preferably mature
individuals whose response to environmental factors is known, and
whose quality traits governed by genotypic and environmental effects
have been identified. For rapid in vitro multiplication of banana, shoot
tips from young suckers of 40100 cm height are most commonly used as
explants.

38.  Media and its combination:
The explant is placed directly on a multiplication inducing culture
medium. For banana micropropagation, MS based media are widely
adopted. Generally, they are supplemented with sucrose as a carbon source
at a concentration of 3040 g/l. Banana tissue cultures often suffer from
excessive blackening caused by oxidation of poly phenolic compounds
released from wounded tissues. These undesirable exudates form a barrier
round the tissue, preventing nutrient uptake and hindering growth.
Therefore, during the first 46 weeks, fresh shoot tips are transferred to new
medium every 12 weeks.
 Stage II: Multiplication of shoot tip cultures:
The formation of multiple shoots and buds is promoted by
supplementing the medium with relatively high concentrations of
cytokinins. In banana, BA is the preferred cytokinin and is usually added in
a concentration of 0.120 mg/l. For the multiplication of propagules, we use
the same medium as for the initiation of shoot cultures (p5 medium
containing 2.25 mg/l BA and 0.175 mg/l IAA).

39.  Stage III: Regenerationof plants:
Individual shoot or shoot clumps are transferred to a nutrient
medium which does not promote further shoot proliferation but
stimulates root formation. The cytokinin in the regeneration medium is
greatly reduced or even completely omitted. Within 2 weeks, shoot tips
develop into un-rooted shoots. To initiate rhizo-genesis IAA, NAA
(naphthalene acetic acid) or IBA (indole3butyric acid) are commonly
included in the medium at between 0.1 and 2 mg/l. We use the same
auxin concentration as in the proliferation medium (0.175 mg/l IAA), but
a tenfold lower BA concentration (0.225 mg/l). For some genotypes
(Musa spp. ABB and BB group) that produce compact proliferating
masses of buds, activated charcoal (0.10.25%) is added to the
regeneration/rooting medium to enhance shoot elongation and rooting.
 Hardening:
After rooting, plants are hardened in vitro for 24 extra weeks on
the regeneration/rooting medium prior to transplantation to soil.

40. Invitro propagationof banana:
Flow chart:
Selection of superior clone
↓
Culture initiation in the production lab
↓
2nd/3rd sub-cultures
↓
Virus-free certified material
↓
Multiplication of shoots up to a maximum of 8 passages
↓
Rooting (in vitro)
↓
Hardening inside the greenhouse, polyhouse & shade area Nursery
↓
Dispatch

41. Tissue culture technique is providing a rapid system for production
of large number of genetically uniform and disease free plantlets for
agriculture and forestry. Although there have been previous reports on date
palm micropropagation through the organogenesis and somatic
embryogenesis the protocols needs to be improved. The objective of the
work is to develop reliable method of organogenesis for date palm.
 Method:
 Plant Material:
The propagation material is used the offshoots were collected. The
selected offshoots were 4-5-year-old, each weighting approximately 6-8 kg.
 Cleaning of explants:
To remove the attached soil and other debris, the offshoots were
washed with the tap water and the outer large leaves and fibers were
carefully removed with the sharp knife until the shoot tip zone was exposed.
Shoot tips were then trimmed to approximately 6-7 cm in length and 4-6 cm
in width.
2. Date palm

42.  Disinfectionand antioxidanttreatment:
The excised shoot tips were washed three-four times with
double distilled water. Thereafter, the cleaned shoot tips were subjected
to two steps of disinfection: a) the washed shoot tips were dipped for 20
minutes in a fungicide (Benlate, 5 g l-1) solution; b) the shoot tips were
dipped in 33% commercial clorox solution for 25-30 minutes. The
explants were then rinsed three times with autoclaved distilled water in
a laminar flow hood. The disinfected explants were then soaked in an
antioxidant solution to minimize oxidation of phenolic compounds
(responsible for the browning of tissues), and to protect them from
desiccation.
 Explant preparation:
After the proper disinfection and antioxidant treatments, the
shoot tips were cut into 1-1.5 cm pieces under the laminar flow hood.

43.  Callusinduction:
Two – four shoot tip pieces were placed on MS medium (Murashige
and Skoog, 1962) solidified with 3% agar-agar and supplemented various
auxins types and concentrations, viz.: 0.0-54.28 μM 2,4-
dichlorophenoxyacetic acid (2,4-D), 0.0-46.96 μM 2,4-5-
trichlorophenoxyacetic acids (2,4,5-T), 0.0-64.31 μM chlorophenoxy-acetic
acid (CPA). 0.3 g l-1 of activated charcoal was added to the medium in
order to remove the phenolic compounds.
 Regenerationand shoot multiplication:
Callus (80-90 mg) was cultured on MS liquid and solid medium
supplemented with beznzylaminopurine (BAP) at concentrations 0.0-8.96
μM and kinetin (KN) at concentrations 0.0-9.28 μM. Data were recorded on
percentage of calli producing shoots (%), shoot number per a callus and
shoot length.
Rooting:
After 14 weeks in the regenerating medium, shoots were separated and
placed on MS liquid and solid medium supplemented with various
concentrations of IBA (0.0-29.52 μM), IAA (0.0-34.24 μM) and NAA (0.0-
32.22 μM).

44.  Mediaand itscombination:
Modified Murashige and Skoog (MS) medium (Table 2) with 3%
sucrose supplemented with 2,4-D (100 mgl-1), NAA (3 mgl-1), 2iP (3 mgl-
1) and kinetin (3 mgl-1) is being successfully used for date palm tissue
culture. pH of the medium should be adjusted to 5.6 and solidified with 8.0
gl-1 of agar. Twenty five ml of medium is dispensed into 40 mm culture
tubes and sterilized for 15 minutes at 121o C. Activated charcoal (1.5 gl-1)
is added to the medium to avoid browning.
 Acclimatizationand hardening:
A sterilized medium comprising peat moss, vermiculite and coarse
sand in the ratio 1:1:1 is ideal for soil transfer. It is recommended to
irrigate immediately with 50% Hoagland solution or 10% MS solution to
avoid dehydration of plants. Containers and potting media must be
adequately cleaned and sterilized. Moisture content of the medium must be
regulated to minimum, and relative humidity of the growth tub must be
maintained high. Acclimatization of date palm is lengthy processes, which
require several subculture steps and careful attention. Otherwise, casualty
rate will be very high. Fifty to ninety percent loss has been reported in date
palm culture during acclimatization.

45. Pomegranate is an economically important fruit crop of the
tropical and subtropical regions of the world that is cultivated for its
delicious fruits. Pomegranate is conventionally propagated by hard wood
and soft wood cuttings. But, this traditional propagation method does not
ensure disease-free and healthy plants. In addition, this method is a very
time-consuming and labor-intensive process. Hence, there is need to
develop an efficient in vitro technique for the propagation of this fruit
trees.
 Method- Shoot tip culture:
 Plant material and explants preparation:
Shoot tip and nodal segments (0.5 - 1.0 cm long) were excised
from two-year-old plants grown in a greenhouse. Following removing of
the leaves, explants were washed with running tap water for 30 minutes,
then were disinfected for 10 min in a 0.1% (w/v) calcium hypochlorite
solution with 2-3 drops of Tween 20 and rinsed three times in autoclaved
distilled water. The sterilized explants (three explants/jar) were vertically
cultured in induction medium.
3. Pomegranate

46.  Culture mediaand culture conditions
Two different media; MS and WPM were used. Sucrose was
added at 30 g/L and myoinstol at 0.1 g/L. The pH of the prepared media
was then adjusted to 5.6 to 5.8 with 0.1 N NaOH, before adding 0.6%
agar.
 In vitrorooting:
Shoot tips with 2-4 cm long developed in vitro were excised and
cultured in half-strength WPM medium containing 50 mg/L myo-
inositol, 15 g/L sucrose and 3 g/L agar. The medium was further
supplemented with 0, 2.5, 4.9 and 9.8 μM IBA or 0, 2.7, 5.4 and 10.8 μM
NAA. After 8 - 10 days, the rooted shoots were transferred to an auxin-
free half-strength medium for further elongation of the roots. Each auxin
treatment consisted of seven replicate jars with one shoot in each. After 4
weeks in culture, number and length of roots per rooted shoot were
evaluated.

47.  Acclimatization and Hardeningof regenerated plantlets:
Well rooted explants were removed from the culture
medium. The roots were washed gently with tap water to remove
agar and then transferred to small plastic pots containing autoclaved
cocopeat-perlite mixture. The pots were covered with polyethylene
bags to maintain high humidity and kept at 25 ± 1°C in artificial
light (50 μmol/m2/s) provided by white fluorescent tubes for 3 to 4
weeks. To harden the plants, polyethylene bags opened gradually,
from a few minutes a day until normal conditions. Plants were then
transferred to larger pots (18 cm diameter) containing garden soil
(soil: compost, 1: 1); kept under shade for another 2 weeks and then
transferred to direct sunlight condition. The survival rate was
examined 40 days after transfer.

48. A retrospective overview of work done on the in vitro shoot tip grafting
in citrus in the world was taken up. In vitro shoot tip grafting is the most
reliable method to recover free citrus saplings from infected parental source.
The technique of in vitro shoot tip grafting is a suitable method for the
elimination of graft transmissible diseases in citrus. Preliminary studies showed
that certain pathogens are difficult to be eliminate from mother plant like citrus
exocortis and stubborn, which might be eliminated by a process of shoot tip
grafting in-vitro. Plant obtained by micro-grafting do not have the same
problems as nucellar plants such as reversion to the juvenile state, excessive
thorniness, vigorous and upright habit of growth, slowness to fruiting, alternate
bearing in early years and physical differences in fruit characteristics.
 Method- In vitroshoot tip grafting in citrus species:
Procedure of in vitro shoot tip grafting: Shoo tip grafting in vitro
consists of grafting, under aseptic conditions with a small shoot tip of 0.1 to 0.2
mm, onto a young etiolated seedling rootstock. The technique has the following
steps: preparation of rootstock, preparation of scion, procedure of grafting,
growing of grafted plants in vitro, and transfer to soil.
4. Citrus

49. Preparation of rootstock forin vitroshoot tip grafting:
Chand L. (2013) conducted an experiment on shoot tip grafting
(STG). They reported that 30-50 per cent frequency of successful grafts was
obtained by using two week old dark grown rootstock seedlings and 0.14 to
0.18 mm long shoot tips as scions. The shoot tip was inserted into an
inverted-T cut made at the top of the decapitated rootstock epicotyls. Most
scion cultivars gave satisfactory grafts on Troyer citrange whereas lemon,
lime and citron yielded successful grafts only on Rough lemon. The age of
the rootstock also has an important influence on grafting success. The
highest rate of successful grafts using Troyer citrange as rootstock was
obtained with two week old seedlings, most shoot tips grafted on younger
seedlings (one week old) were covered with precocious callus formation by
the rootstock, whereas most shoot tips grafting on rootstock.
The scion taken from in vitro cultured buds produced healthy plants
than scions from whole tree. The frequency of successful grafts increased
with the size of the shoot tips but the per cent of virus free plants declined,
although this depended on the pathogen. In citrus, the shoot terminals
excised from mature bearing trees, actively growing shoot flushes from field
or greenhouse.

50. Special problems encountered by in vitro culture:
Hyperhydricity:
Hyperhydricity (originally called vitrification) is characterized by a
translucent, water-soaked, succulent appearance that can result in cultures that
deteriorate and fail to proliferate. Physiologically, expression involves excess
water uptake : (“waterlogging”) and inhibition of lignin and cellulose
synthesis. Hyperhydricity appears to be a consequence of the difference
between the water potential in the medium and developing shoots, as well as
a low nitrate to ammonium ratio. Hyperhydricity can also be caused by
hormonal imbalances, especially ethylene.
Internal Pathogens:
Even though micropropagation is considered to be a pathogen-free
system, complete absence of pathogens should not be taken for granted. The
original source material should be indexed for specific viruses even if the
original selection is from shoot-tip cultures or micrografts.
For example, in the initial enthusiasm for orchid micropropagation, the
importance of initial meristem treatment was not appreciated and shoot-tip
culture was assumed to control viruses. As a result, many of the commercial
sources of orchid cultivars were soon found to be infected, causing
considerable economic loss.

51. Excessive Exudation:
One of the most frequently encountered problems with
establishing explants (especially woody perennials) in culture is the
production of exudates by the explants. The exudates are usually
considered to be various phenolic compounds that oxidize to form a
brown material in the medium that tends to be inhibitory to
development. Treatments to minimize this problem include treating the
explant with an antioxidant (citric or ascorbic acid), including an
adsorbent material in the medium (polyvinylpyrrolidone or activated
charcoal), and frequent transfers to a new medium.
Shoot-Tip Necrosis:
Actively growing shoot tips sometimes develop “tip die- back”
usually caused by calcium deficiency that can be corrected by refining
the basic nutrient medium to include more calcium

52. Tissue Proliferation:
Tissue proliferation (TP) is the formation of gall-like
growths on the stem of micropropagated plants (146). It may
occur while plants are still in culture or may not be seen until
after plants have been moved to the greenhouse or nursery.
Habituation:
Habituation is the autotrophic growth in cultures that
had previously required auxin or cytokinin for growth. For
example, shoot cultures may become habituated to cytokinin
and continue to proliferate even after the culture has been
transferred to a medium without any growth regulator.