Micropropagation.

Propagation of plants refers to the production of a new individual plant. The propagation of the plant can be achieved by either sexual or asexual mode.

• Sexual mode of propagation:

Sexual mode of propagation is through the production of seeds and involves sexual reproduction (fig 1). The progeny receives genetic material from both of the parent plant, hence resulting into seedling variability.

• Asexual mode of propagation:

In the asexual mode of propagation, the vegetatively active part from a single parent plant is used to generate new individual plants. The new plants thus generated will be genetically similar or ‘clones‘ of the parent plant. Hence the progeny would be uniform and maintain the desirable characteristics of the parent. The different methods used in asexual propagation are cuttings, layering, budding, grafting (fig 2) and micropropagation.

~ Micropropagation:

Micropropagation is a plant tissue culture technique which can be defined as:

‘A type of asexual propagation technique in which the plant material is grown in aseptic condition on nutrient media to produce a large number of plants with same characteristics as the mother plant’.

Like any other asexual mode of propagation, micropropagation too, produces genetically identical plants.

This tissue culture technique has a few basal requirements:-

i. Mother plant:

The mother plant is the plant which is to be propogated. It should have a desirable characteristic, such as good quality fruits, high growth rate, high secondary metabolite content, etc. This plant should be healthy and disease-free.

The explant is small part of the mother plant which excised and used for the tissue culture procedures. Theoretically any part of the plant can be used as explant, as the plant cells are considered to be *totipotent. Hence the explant can range from shoot tips, leaves, nodal segments, internodal segments, anthers, embryos, pollen grains, etc.

(*Totipotency is the ability of a cell to give rise to a whole plant. In plants, all the cells, including the matured and differentiated cells, possess the ability to become meristematic, and hence totipotent. This needs the differentiated cell to first undergo ‘dedifferentiation’ and then ‘redifferentiation’.)

ii. Hormones:

Along with basal nutrient media, de novo shoot meristem formation requires increased levels of auxins and cytokinins. Both these hormones are involved in the cell division and meristem establishment and activity.

However, in general,

-shoot formation is favoured by higher amount of cytokinin

-higher auxin concentrations favours rooting

-both the hormones in around equal amount leads to callus formation (see fig 3).

(Just for info: Need to know on plant hormones??)

iii. Growth conditions:

The tissue culture procedures needs an aseptic environment. It is usually achieved by using laminar air filter and the plants are incubated in a specially designed growth chamber (fig 4). This growth chamber is usually set at 20–24 °C. The light intensity ranges from 2000 to 4000 lux with the photoperiod (light) of about 12 to 16 hours.

• Different techniques in Micropropagation:

There are different techniques used for the regeneration of plants from an explant, namely:

1. Direct Regeneration:

Direct regeneration refers to the generation of shoots directly from the explant, without the formation of callus. Based on the explants, the shoots regenerated can be from apical/axillary meristematic tissues or other explants (adventitious).

• Shoots from Axillary/ Apical meristematic tissues:

The apical and axillary buds of the plants (fig5) are structures developing from the meristematic tissue. The meristematic tissues are group of small cells with dense cytoplasm and large nuclei (similar to stem cells in animal kingdom).

The apical and axillary buds are capable of producing shoots.

These explants when used for the in vitro regeneration with proper hormone combination of cytokinins like kinetin and benzyladenine, can directly give rise to a large number of shoots (fig 6 & 7).

The shoots are then trimmed and transferred to fresh medium for further proliferation or can be singled and transferred to rooting media.

(Just for info: Read a paper on direct regeneration using shoot tip explant in lavender)

• Adventitious shoots:

Adventitious means in an unusual anatomical position. Hence, the term ‘adventitious shoots’ mean the shoots are grown from the place where they are not formed normally. The explants used for adventitious shoot induction include internodes, leaf blades, cotyledons, root tips, bulb, corms, tubers, rhizomes and so on.

The proper hormonal combinations result in de novo meristematic zone formation i.e. ‘dedifferentiation’. The shoots are then induced and later proliferated in the appropriate hormone combination.

The shoots obtained can be trimmed and again proliferated on fresh media with proper hormones or singled out and allowed to root.

(Just for info: Here’s a paper on Direct regeneration of eggplant from leaf segments.)

2. Somatic embryogenesis:

Somatic embryogenesis (SE) is a means by which plants can regenerate embryo from a somatic cell. In presence of the optimum hormonal combination the explants, expression of gene is modified, giving rise to a bipolar embryo-like structure (fig 8).

Different plant growth regulators (PGR) including auxins, cytokinins, ethylene and abscisic acid are used together for the induction of SE. The somatic embryoids are diploid and grow into new plantlets, hence suited for clonal propagation.

(Just for info: Read the paper on SE titled ‘Signaling Overview of Plant Somatic Embryogenesis’.)

3. Indirect regeneration:

In the indirect regeneration, first the callus is induced from the explant, which is an unorganized mass of actively dividing cells. The shoots or embryo are then induced from the cells of the callus. Each step requires different combination of hormones.

The cells of callus are not geneti­cally stable and may result in heterogeneous group of next generation plants.

(Just for info: Here’s a paper on callus induction and indirect plant regeneration from various tissues of Jatropha curcas.)

Hence, direct plantlet formation is more desirable in case of micro- propagation or clonal propagation.

~The process of micro-propagation can be divided into five different stages, which we shall discuss in the next post.

—-

This is all for this post. Hope u like this post, if yes please comment, like and share!!

Also follow us on Facebook, Twitter, Instagram or mail us (thebiotechnotes@gmail.com).

Have a nice day!

Thank you!!

—-

Read other posts by The Biotech Notes:

Bacteriophage Reproduction: Lysogenic Cycle.

Chromosome Banding..

DNA Replication: Prokaryotes.

—-

References:

-Goldberg et al. (1994) Plant embryogenesis: zygote to seed. Science 266(5185):605-14.

-Faleiro et al (2019). Advances in passion fruit (Passiflora spp.) propagation. Revista Brasileira de Fruticultura 41(2): (e-155).

-George et al. (2008) Plant Growth Regulators II: Cytokinins, their Analogues and Antagonists. Plant Propagation by Tissue Culture. Springer, Dordrecht 205-226.

-Sathish (2018) Efficient direct plant regeneration from immature leaf roll explants of sugarcane (Saccharum officinarum L.) using polyamines and assessment of genetic fidelity by SCoT markers. In Vitro Cellular & Developmental Biology – Plant 54 (4): 399–412.

-Husain et al., (2010) Somatic embryogenesis and plant regeneration in Pterocarpus marsupium Roxb.. Trees. 24 (4): 781-787.

-Mehaboob et al. (2019). Effect of nitrogen sources and 2, 4-D treatment on indirect regeneration of ginger ( Zingiber officinale Rosc.) using leaf base explants. Journal of Plant Biotechnology. 46. 17-21.

–Raven, Peter H. & Johnson, George B. (2002). Biology (6th ed). McGraw-Hill, Boston.

-Méndez-Hernández et al. (2019) Signaling Overview of Plant Somatic Embryogenesis. Front Plant Sci.10: 77. Published online 2019 Feb 7.

-Bhojwani & Razdan (1996) Chapter 5 Cellular totipotency. Studies in Plant Science 5: 95-123.

-Hill & Schaller (2013) Enhancing plant regeneration in tissue culture, Plant Signaling & Behavior 8:10, e25709.

-Mir et al. (2013). Fast and Efficient In-vitro Multiplication of Apple Clonal Root Stock MM-106. Vegetos- An International Journal of Plant Research 26(2): 198-202.