Bridg, Hannia: Micropropagation and Determination of the in vitro Stability of Annona cherimola Mill. and Annona muricata L.


Chapter 3. Micropropagation and Genetic Stability

3.1 Micropropagation

The in vitro plant cell and tissue culture is defined as the capability to regenerate and propagate plants from single cells, tissues and organs under sterile conditions and controlled environmental conditions (Murashige and Skoog, 1974).

The in vitro culture is often used as model system in the study of various physiological, biochemical, genetic and structural problems related to plants. There are several ways to promote the in vitro regeneration of a selected plant material relying on the initial material (Torres, 1989):

Plant culture \|[xrArr ]\| From seedlings or larger plants

Embryo culture \|[xrArr ]\| From isolated mature or immature embryos

Organ culture \|[xrArr ]\| From isolated plant organs

Tissue culture or Callus culture \|[xrArr ]\| From tissues arising from
explants of plant organs

Suspension culture or Cell culture \|[xrArr ]\| From culture of isolated cells or
very small aggregates remaining
dispersed in liquid medium

Protoplast culture \|[xrArr ]\| Culture from protoplats, i.e., cells devoid of their retaining walls

Anther culture or Haploid culture \|[xrArr ]\| Micropropagation of anthers and/or immature polen grain in an effort to obtain a haploid cell or callus line

According to the objectives to search for: mineral salt combinations, plant growth regulators and environmental factors are variables which can be manipulated to promote organogenesis, differentiation and dedifferentiation of the selected initial cultured explant (Torres, 1989 ; de Fossard, 1977). Plant tissue culture techniques have a great potential as a means of vegetatively propagating economically important crops and crops of future potential on a commercial basis (Roca and Mrogiski, 1991).

Micropropagation is a vegetative multiplication system based on the promotion of growth of microcuttings from axillary buds of apically dominant plants, growth of shoots from nodal sections, dissection of axillary shoots on rooting medium and production of small rooted shoots (Drew, 1997).

Micropropagation has been defined as in vitro regeneration of plants from organs, tissues, cells or protoplast (Beversdorf, 1990) and the true-to-type propagation of a selected genotype using in vitro culture techniques (Deberg and Read, 1991).


As shown in the Table 15 the promotion of in vitro plants is divided in stages (Murashige, 1974), which describe not only the micropropagation steps but also identify the morphogenic events of the selected explant or initial culture plant material in aseptic conditions.

Table 15. Micropropagation steps










Stock Plant Selection and Adaptation

Reduction of endogenous tissue contaminants

Definition of the stock or mother plant and source preparation of explants



Promotion of in vitro aseptic culture

Explant selection, elimination of exogenous contaminants and new in vitro adaptation



Somatic cell embryogenesis

Promotion of In vitro cell or tissue organogenesis and rapid



Enhanced axillary development

differentiation-multiplication of new shoots



Adventitious shoot development




Promotion of in vitro plant regenerants

The rhizogenesis of the new in vitro derived shoots is stimulated under ex vitro or in vitro conditions



Stimulation of autotrophic metabolism

Hardening of vegetative structures



Improvement of the normal development of an in vitro regenerant

Open growth of regenerants

3.1.1 Application

Micropropagation has also been useful for the rapid initial release of new varieties prior to multiplication by conventional methods, e.g. pineapple (Drew, 1980) and strawberry (Smith and Drew, 1990).

Micropropagation is also used to promote germplasm storage for maintenance of disease-free stock in controlled environmental conditions (Withers, 1988) and in long term via cryopreservation (Kartha, 1985).

This type of in vitro vegetative propagation has important benefits to produce stable lines in plants that have no named varieties e.g., Annona spp. (Bridg, 1993b), Australian dioecious papaw genotypes (Drew, 1988; Drew, 1992) where traditional plant breeding has failed. It has also a great potential for the propagation of important crops like: Cassava spp., Oryza spp., Phaseoulus spp., Solanum spp., principally (Roca and Mroginski, 1991). The micropropagation of elite or selected plants showed good results which benefit the agriculture, horticulture and forestry (Conger, 1981; Drew, 1997).


Worldwide there is much interest to promote the development of an in vitro technology that permits the propagation and breeding of commercial valuable woody, semiwoody, ornamental, basic food, industrial and medicinal plants. These species are in danger of extiction and should receive a priority in terms of gemplasm conservation (Conger, 1981; Deberg and Zimmerman, 1990; Drew, 1997).

3.1.2 Limitation

In Plant Tissue Culture the genetic stability of the in vitro produced plants is cause of preocupation when clonal micropropagation is applied in terms of conservation and incrementation of germplasm lines. The in vitro promotion of clonal true-to-type plants is a priority, as result of the report of true-off-type plants comming from in vitro culture (Skirvin, 1978).

When an in vitro clone multiplication system introduces a high risk of genetic variation, it is not desirable. The culture-derived plants have to represent genetically the source of material (Withers, 1989) and failures to understand this principle have resulted in disastrous consequences for some species (Smith and Drew, 1990b; Beversdorf, 1990).

There are numerous reviews that describe the variability observed on in vitro plant regenerants (Reisch, 1983; Evans, 1984; Evans et al. 1984). This changes are in vitro regenerants with a new genome modifications as the result of culture media traces that are interferring the plant genome e.g., chimeras or genetic off-types (Krikorian et al. 1983; Tilney-Bassett, 1986; Torres, 1989).

The ability to propagate in vitro free-off plants or genetic off-types is dependent on the technique used during the micropropagation (Pierik, 1987).The tissue culture variability could be a directly generated effect of several and interacting factors on the multiplied cells (Karp and Bright, 1985; Vuylsteke et al. 1988; Meier et al. 1988).

Murashige (1974) has described three types of in vitro culture differentiation, which have been defined earlier for cultures of higher plants and are dependent of the cell-tissue organization of the initial explant:

I. Organized tissue


II. Non Organized tissue

III. Combination of non-oganized and oganized tissue

Protocols for in vitro plantlet regeneration via organogenesis could be the basis of the development of a transformation system applied on Annona spp. Molecular markers would be useful both for documenting genetic diversity and elucidating the confusion on the name and origin of many commercial cultivars (Jordan and Botti, 1992; Drew, 1997).

3.2 Somaclonal Variation

The term somaclonal variation has been used by Larkin and Scowcroft (1981) and refers to the phenotypic and genotypic variation observed in regenerated plants from any form of cell culture.

When an explant, any plant segment, is subjected to a tissue culture cycle it might be possible to obtain a somaclonal variant (D‘Amato, 1978, Rani et al. 1995, Hashmi et al. 1997). This cycle includes establishment of a dedifferentiated cell or tissue under


defined conditions and subsequent organogenesis and regeneration of plants (Hammerschlag, 1992). The genetic variation has been shown to originate either from the original explant or from exposure to a tissue culture cycle (Skirvin and Janik, 1976) and is often heritable (Larkin et al. 1984; Breiman et al. 1987).

3.2.1 Factors

The source of somaclonal variation has not been completely explained and the degree and frequency of somatic genetic changes in vitro is uncertain (Phillip et al. 1990). Variation, as has been shown, is conditioned by source of material, plant genotype, kind of explant, cell segregation of primary explant, development, organogenesis (D‘ Amato, 1975; Drew, 1997) and number of subcultures (Skirvin and Janik, 1976). All of these factors are correlated with time of culture and culture media (D‘Amato, 1964; Evans and Gamborg, 1982; Dolezel and Novak, 1984; Scowcroft et al. 1987; Schilde-Rentschler and Roca, 1987).

The importance of culture medium and particularly the exogenous application of plant growth regulators (PGRs) has been discussed (Terzi and LoSchiavo, 1990). The embryogenic potential of explants is closely associated with the content of PGRs, the balance and concentration among naturally produced and exogenous applied PGRs.

Due to the complexity and sophistication of the techniques required for PGRs analysis full recognition of these techniques is still in process. Only a few research teams have found links between the embryogenic capacity of plant tissues and a specific endogenous hormonal content (Rajaserakan et al. 1987a, b; Wenck et al. 1988; Ivanova et al. 1994).

Cytokinins required for shoot induction and multiplication may cause abnormalities such as decreased rooting, stunted or compact plants, increased branching or slender stems and leaves (Karp and Bright, 1985). Albinism is an abnormality that often occurs during in vitro propagation. The albino plants might be due to the dis-organization of the ribosomal RNAs and plastids in vitro and (Torres, 1989).

Abnormalities in regenerated plants have been less frequently attributed to auxins (Smith, 1979) as abnormal fertility of the flowers, abnormal petal shapes, decreased vigor, malformed leaves, and increased lateral shoot formation (Bilkey and Cocking, 1981). Other factors that may result in the production of abnormal plants include Gibberellic acid (GA3), temperature, osmotic potential of the medium and agar or gelrite concentration (Torres, 1989).

3.2.2 Phenotypic changes

Phenotypic variation is expressed principally by changes in morphological characteristics, which can be affected for instance by growth habit, plant height, shoot length, number of primary branches, flower quality, fruit colour, production, uniformity, disease resistance and tolerance to environmental conditions (Skirvin, 1978; Reisch and Bingham, 1981; Evans et al. 1984; Hammerschlag, 1992). This variation in plant regenerates has been reviewed in most of the cases for some herbaceous plants, but not for woody species (Economou et al. 1981).


Some phenotypic changes observed in in vitro derived plants include the presence or absence of pubescence, changes in leaf morphology, dwarfs, loss of pigmentation, and alteration in flower morphology. Reduction or enhancement in plant vigour may also occur in plants propagated through tissue culture. Many of these abnormalities appear to have an epigenetic or physiological basis and are therefore reversible. Variegation in leaves may appear in certain species and disappear in others when plants are propagated in vitro (Torres, 1989).

3.2.3 Genetic changes

Somaclonal variation due to karyotypic changes is a widespread phenomenon in plant cell culture, and it might affect plant breeding (Larking and Scowcroft, 1981; Wenzel, 1985). Somaclonal variants in some cases seem to be similar in magnitude to regenerates from cells exposed to mutagenic agents.

Although a number of different types of genetic mutations has been described as the basis for somaclonal variation (Larkin and Scowcroft, 1981), the recognition of genetic and epigenetic changes that occurs during in vitro culture and on plants derived from these cultures actually remains to be discussed (Reish, 1983; Krikorian, 1983; Roca and Mroginski, 1991).

New genome arrangements, aneuploids not separable in culture, mitotic break off that conduces to polyploid lines, genome reorganisation by transposoms, amplification or reduction of genes, effects of inversion, trans-location, changes in chromosome number, chromosomal rearrangements, karyotype variation, prevalence of specific plant karyotype in polysomatic plants or tissues, alterations of the interaction nucleus-cytoplasm have been demonstrated as basis of somaclonal variation (Larkin and Scowcroft, 1981; Karp et al. 1982; McCoy et al. 1982; Orton, 1983).

3.2.4 Screening

The genetic stability or fidelity adviser of in vitro produced crop plants are gaining importance (Larkin and Scrowfort, 1981; Goto et al. 1998) with special emphasis on chromosome arrangements (D‘Amato, 1978; Whiters, 1989).

The characterization of clone regenerants comming from in vitro culture by traditional methods such as morphological descriptions, physiological supervisions and cytological studies have been made. These methods are time-consuming processes based on characters which can be affected by the in vitro manipulation and it is no easy to differentiate clonal selections or to predict genetic identity with a high probability (Patel and Berlyn, 1982, Meier, 1982, Mo et al. 1989).

Moreover, biochemical molecular analysis by isoenzymes and electrophoresis techniques to separate molecules have been developed in order to detect genome modifications (Renfroe and Berlyn, 1984; Mo et al. 1989; Shenoy and Vasil, 1992). However, these analytical approaches have serious limitations because are describing only partially the genetic changes and there are difficulties in assess those changes if any, at the DNA sequence level. In addition the evaluation and interpretation of the results cover many years of research and probes are time dependent. (Rani et al. 1995; Rani and Raina, 1998).


The DNA molecule maintains its characteristics permanently because it is not conditionated by the time, irrespectively of its source. The molecular DNA technology by the genetic markers is comparatively less complex, quick to perform and requires only a small amount of probe material. Furthermore DNA extraction protocols are simple, suitable and cover a wide spectrum (Rafalski et al. 1993a)

Recently several DNA molecular techniques such as Restriction Fragment Length Polymorphic DNA (RFLP) (Shirzadegan et al. 1991), Randomly Amplified Polymorphic DNA (RAPD) (Rani et al. 1995), DNA Amplification Fingerprinting (DAF) (Vos, 1995) and Arbitrarily Primed PCR (AP) (Vos et al. 1993) have been introduced and offer several advantages compared with the cytological, biochemical and physiological methods, where the fundamental and practical questions of plant tissue culture remain unsolved (Williams et al. 1990).

The application of DNA molecular markers in genetic mapping, genetic diagnostics, molecular taxonomy and evolutionary studies has been established since 15 years ago, nevertheless DNA polymorphisms origins and causes are still in study (Vos et al. 1995).

3.3 Random Amplified Polymorphic DNA

Detection of DNA markers can be based either on Southern hybridisation or Polymerase Chain Reaction (PCR) amplification techniques. In Southern hybridisation the detection of a fragment is based on sequence homology with the DNA probe. Therefore, this method enables the detection of homologous DNA sequences among distantly related species using the same DNA probe. This has applications in research on genome evolution. Otherwise the DNA fragments detection by Southern hybridization is laborious and incompatible with applications which requires a high resolution.

The most commonly used DNA markers are RFLP (Restriction Fragment Length Polymorphisms) because of its reliability and the possibility of detecting multiple alleles of the same locus. The utility of DNA markers for genetic mapping has enabled the location of several genes of agronomic interest within the context of an existing RFLP Polymorphic map (Rafelsky et al. 1993).

The PCR recognition of specific sequences by the PCR-primers allows for very little sequence redundancy, therefore, PCR-based markers are generally highly species-specific (Vos et al. 1995) Among PCR-based marker technologies two main types of techniques may be distinguished, targeted and random amplification techniques.

The random amplification techniques rely on PCR techniques using arbitrary primers at low annealing temperatures. The amplification products are the primer binding sites. Fragment patterns cannot be predicted, but are primer specific. All these random DNA polymorphisms are generally detected as presence/absence of amplified fragments. These marker techniques are known as mono-allelic DNA markers (Vos et al. 1995).

Genetic tests based on PCR (Polymerase Chain Reaction) are simple to perform, but target DNA sequence information is required to design specific primers. The Random Amplified Polymorphic DNA markers (RAPD), have recently been applied in woody species (Goto et al. 1998) to asses the reproduction of some segments of the genome, as rapid appraisal of tissue-culture-derived plants (Rani and Raina, 1998). They


have been shown to enhance breeding efforts in annual and perennial crops (Rafelski and Tingey, 1993). They are also effective for cultivar identification (Ronning et al. 1995b).

The DNA amplification with RAPD not requires previous knowledge of natural target DNA sequence. The amplification of random DNA segments is made with single primers (usually 10-mers) of arbitrary nucleotide sequence (Williams et al. 1990). The assay is not radioactive, requires only nanogram quantities of DNA and is applicable to a broad range of species.

To perform a RAPD assay a single oligonucleotide of an arbitrary DNA sequence is mixed with genomic DNA in presence of a thermostable DNA polymerase mixed with a suitable buffer and then is subjected to temperature cycling conditions “polymerase chain reaction“. The products of the reaction depend on the sequence and length of the oligonucleotide, as well as the reaction conditions.

At an appropriate annealing temperature during the thermal cyle the simple primer binds to sites on opposite strands of the genomic DNA that are within an amplifiable distance of each other and a discrete DNA segment is produced. Allelic variation among individuals is detected as the presence or absence of the multiplication product visualized as a band after PCR and electrophoresis (Rafalski et al. 1993b).

The DNA amplification reaction is repeated on a set of DNA samples with several different primers, under conditions that result in several amplified bands from each primer. The presence or absence of this specific product, although amplified with an arbitrary primer, will be diagnostic for the oligonucleotide-binding sites on the genomic DNA. Polymorphic bands are noted, and the polymorphisms can be mapped in a segregating population. Often a single primer can be used to identify several polymorphisms, each of which maps to a different locus.

Analysis of variations at the nuclear genome level using RAPD has advantages over RFLP as a single primer produces several loci, covering a larger portion of the genome.

According to Jayasankar et al. (1998) RAPD have been used to determine:

Genetic relationships in Annona spp. (Ronning et al. 1995), Theobroma cacao (Ronning et al. 1995) and Carica papaya (Drew, 1988).

To determine phylogenetic relationships in Mangifera indica (Schnell and Knight, 1993)

Cultivar identification (Ronning et al. 1995)

Confirming somatic hybrids following protoplast fusion between Citrus spp. (Deng et al. 1995)

Somaclonal variation in regenerants from protoplast, and from embryogenic cultures derived from zygotic embryos in Vitis spp. (Schneider et al. 1996)

To identify resistant gene in near-isogenic tomato lines linked to a bacterial resistant (Martin et al. 1991)

To identify selected resistant lines from embryogenic Mangifera indica cultures for resistance to Colletotichum gloesporoioides (Jayasankar et al. 1998)

For index Avocado viroid species (Ronning et al. 1997).

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