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Techniques of In vitro clonal propagation for fruit crops

Pawan Kumar NagarM.Sc. (Horti.) Fruit science

REG. NO: 04-2690-2015

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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 fruit crops

Commercial Tissue culture propagation is particularly useful in the following situations:

Where the propagation rate is slow by conven tional means New cultivars with high market demand Cultivars with high market value Plants which is difficult to propagate clonally Conservation of endangered species.

Disadvantages of Tissue culture propagation:

High labor costs A requirement for expensive and sophisticated facil ities, trained

personnel, and specialized techniques A high-volume, more or less continuous distribu tion system, or

adequate storage facilities to stock pile 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

General laboratory facilities and procedures:

Facilities and 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.

Media Preparation: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 saltsOrganic compoundsGelling agents

There are basically four stages to the micropropagation process.Stage I. EstablishmentStage II. Shoot multiplicationStage III. Root formationStage IV. Acclimatization

Micropropagation procedures:

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.

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 usedSome 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.

Establishment stage

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.

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.

Root formation stage

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.

Acclimatization

Types of tissue culture systemsThe procedures used for tissue culture that are associated with propagation. These include the formation of:

1. Plantlets2. Seedlings3. Callus4. 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.

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: EstablishmentStage II: Shoot multiplicationStage III: Root formationStage IV: Acclimatization

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

In vitro techniques of clonal propagation (Tissue culture):

Shoot-tip micrografting in vitroGrafting 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

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

Shoot tip micrografting in vitro

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

Guha and Maheshwari (1964)

Anther culture

Embryo Culture and Embryo Rescue: 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.

Embryo rescue or embryo culture

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.

Ovary and ovule culture

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

Callus Formation

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.

Cell suspension culture

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.

In vitro Techniques of

Propagation of Fruit Crops:

1.Banana: Method: Shoot-tip cultures:

Banana is commercially propagated by shoot tip culture in vitro.

Stage I: Initiation of 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.

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).

Stage III: Regeneration of 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.

In vitro propagation of 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

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

Disinfection and antioxidant treatment:

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.

Callus induction: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. Regeneration and 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).

Media and its combination: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. Acclimatization and 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.

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

Culture media and culture conditionsTwo 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 vitro rooting: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.

Acclimatization and Hardening of 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.

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 vitro shoot 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

Preparation of rootstock for in vitro shoot 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.

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.

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

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.