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  Section: General Biotechnology / Plant Biotechnology
 
 
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In Vitro Culture Techniques : The Biotechnological Principles

 
     
 
Shoot Culture and Micropropagation
In shoot culture, apical meristem (the region of shoot apex laying distal to leaf primordium) is cultured. It clearly differs from shoot apex by having shoot apex and a few leaf primordia. Because of culture of relatively large tips (5-10 mm long sections), this technique is also known as meristem culture, meristemming and mericlones (Murashige, 1974). Shoot tip culture is extensively applied in horticulture, agriculture and forestry. Morel (1960) pioneered the work of shoot apex culture of orchid, Cymbidium, for the clonal multiplication. Among the workers Murashige significantly contributed for establishing micropropagation technique and its further biotechnological application. Because of minute size of the propagules in culture, the in vitro propagation technique is known as micropopagation.

Murashige (1974) has described the procedure of development of micropropagation into three different developmental stages: Stage I, establishment of explant aseptically; stage II, multiplication of propagules by repeated subcultures on a specific nutrient medium; and stage III, rooting and hardening of plantlets and planting into soil.

Fossard (1987) have describe the following tour stages of micropropagation:

Stage I : Selection of a suitable explant and inoculation into nutrient medium;

Stage II : Multiplication and growth of culture which takes about 2 months, followed by repeated subculture. At this stage rootless (with apical dominance, AD) cultures are obtained. In contrast, in fast multiplying cultures rootless multishooted (MS) cultures are also developed;

Stage III: At this stage cultures are obtained by changing the medium to planting out in the following 3 substages:

(a)

Microcutting Stage (MC): In this case, culture is transferred to multishoot inducing medium as to get longer shoot and to harvest by MC. Finally MC is subcultured on medium for rooting. Stage II AD cultures also respond to MC procedure;

(b)

Stage-III MS Culture Stage: At this stage, stage II MS cultures are divided into individual shoots which are induced to form apical dominant shoots with roots;

(c)

MS Culture Stage: Small clumps of culture from stage II MS culture are transferred to AD inducing medium to get a bushy plant. Generally this type of plant is preferred for many ornamental horticulture plants;

 

 

Content

Totipotency

Historical background

Requirements for cell and Tissue Cultures

 

A tissues culture laboratory

 

Nutrient media

 

 

Inorganic chemicals

 

 

Growth hormones

 

 

Organic constitutents

 

 

Vitamins

 

 

Amino acids

Culture of plant materials

 

Explant culture

 

Callus formation and its culture

 

Organogenesis

 

Root culture

 

Shoot culture and micropropagation

 

Cell culture

 

 

Benefits from cell culture

 

Somatic embryogenesis

 

Somaclonal variation

 

Protoplast culture

 

 

Isolation

 

 

Regeneration

 

Protoplast fusion and somatic hybridization

 

 

Fusion products

 

 

Method of somatic hybridization

 

Anther and pollen Culture

 

 

Culturing techniques

 

In vitro androgenesis (direct and indirect androgenesis)

 

Mentor pollen technology

 

Embryo culture

 

Embryo rescue

 

Protoplast fusion in fungi

Stage IV: This is the planting out stage where plantlets are aseptically removed from test tube environment to natural and harsh environment. At this stage roots should be fully functional in potting mix (the soil environmental where plantlets are transplanted). During this procedure generally plantlets fail to survive because of desiccation (from 100% humidity of test tubes to low humidity under ambient conditions), harsh environment, invasion of soil microorganisms, unadjustibility from dependent (artificial medium) to independent nutrition (by photosynthesis).

These procedures generate the concept that a single medium is not sufficient for multiplication and regeneration of plants. Establishment of explant is influenced by the tissue of explants, constituents of nutrients and environmental factors. The region of young plant having cells (shoot tip, auxiliary buds) is the suitable explant.

Mature tissue impregnated with high amount of phenolic compounds is difficult to culture. Therefore, oxidation of these phenolics is necessary before culturing them. Hu and Wang (1983) have suggested for (i) adding antioxidants to the medium, (ii) pre-soaking of explants in antioxidant solutions before culture, (iii) sub-culturing to a fresh medium, and (iv) providing light or no light during the start of culture.

In addition to nutritional composition, light and temperature are equally important in propagation. For morphogenesis and biosynthesis of chlorophylls, fluroscent light is necessary. For stage I and II light intensity of 1,000 lux has been found best, and for stage III it was recorded between 3,000-10,000 lux (Murashige, 1974).

A medium that does not initiate callus formation is thought to be suitable because the genetic instability of callus will lead to a high degree of genetically aberrant plants (Dodds and Roberts, 1985). Chemical constituent that favor callus development should be avoided. The source of auxin is usually IAA, a-NAA and IBA (indole-3-butyric acid). The auxin and 2, 4-D should be discarded as they stimulate callus formation and inhibit organogenesis. Exogenous GA3 (0.1 ml/ liter) is required for the development of shoot tips of isolated potato, carnation, Chrysanthemum and Dahlia and their subsequent micro-propagation (Morel, 1975).

At planting out stage, it is necessary to develop acclimatization capability in plantlets before removing them from the test tubes. This can be done by (i) induction to develop some normal and functional leaves, (ii) induction of functional roots, and (iii) exposing the in vitro cultures to harsh environment before first two weeks of planting out. Fossard (1987) has described the successful potting mix as peat alone, vermiculite alone, mixture of loam-peat, peat-perlite-vermiculite-ash, perlite-pulverized pine bark-peat-river sand, and perlite-vermiculite-sand (see Section 9.2.1).

  Morphogenetic responses of shoot and root explants of tropical trees. Syzygium cutnuni (A-B); A -shoot formation from the nodal segment excised from seedlings after 2 weeks of culture; B-multiple shoot formation after 4 weeks of culture. Morus nigra (C-G); C -rooting of shoots on MS + NAA (1.0 mg/1) medium after 3 weeks of subculture; D-establishment of plantlets into soil after one month of transfer; E-female inflorescence formation after 2, 3 and 4 weeks (on MS + BAP (1.0 mg/1) medium from mature nodal segments; F-female inflorescence formation on MS + BAP (1.0 mg/1) + IBA (0.5 mg/1) after 4 weeks; G-complete plantlet with ripen fruits after 8 weeks of subculture (on MS + BAP + IBA medium. (Courtesy : Dr. Usha Yadava, B.H.U., Varanasi).
 

Fig. 8.3. Morphogenetic responses of shoot and root explants of tropical trees. Syzygium cutnuni (A-B); A -shoot formation from the nodal segment excised from seedlings after 2 weeks of culture; B-multiple shoot formation after 4 weeks of culture. Morus nigra (C-G); C -rooting of shoots on MS + NAA (1.0 mg/1) medium after 3 weeks of subculture; D-establishment of plantlets into soil after one month of transfer; E-female inflorescence formation after 2, 3 and 4 weeks (on MS + BAP (1.0 mg/1) medium from mature nodal segments; F-female inflorescence formation on MS + BAP (1.0 mg/1) + IBA (0.5 mg/1) after 4 weeks; G-complete plantlet with ripen fruits after 8 weeks of subculture (on MS + BAP + IBA medium.

Yadav at al. (1990 a,b) successfully cultured shoot tips and nodal explants of two tropical trees (e.g. Morus nigra and Syzygium cuminii) in vitro. From these explants multiple shoots and roots were proliferated by providing different nutrient conditions. Finally the regenerated plants were transferred into the soil. In a separate experiment, inflorescence (in S. cumminii) and fruits (in M.nigra) were also developed successfully in vitro (Fig. 8.3) (Yadav, 1990).
 
     
 
 
     



     
 
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