Applicazioni Colture in vitro Produzione in tempi relativamente breve di

un grandissimo numero di piante geneticamente identiche

Ottenimento di piante virus-free

Ottenimento di piante geneticamente modificate

Conservazione del germoplasma

Ottenimento di ibridi somatici-fusione di protoplasti

Produzione di metaboliti secondari

Produzione di semi artificiali

Micropropagation and Regeneration

Adventitious Regeneration

Such freshly originated organs are said to be adventive or adventitious

New organs such as shoots, roots or embryos can be induced to form on plant tissues lacking preexisting meristems.

Classification of Adventitious Regeneration

Caulogenesis: shoots

Rhizogenesis : roots

Somatic embryogenesis: embryos

MORPHOGENESIS

Caulogenesis

Rhizogenesis

Adventitious meristems can theoretically occur in either

of two distinct ways.

Directly from the differentiated cells in a

newly transferred piece of whole-plant tissue, without intermediate proliferation of undifferentiated tissue

Indirectly from the unspecialised, unorganised and dedifferentiated cells

of callus tissues or suspension cultures.

Two types of adventitious regeneration

Organogenesis:

Caulogenesis

Rhizogenesis

Somatic embryogenesis:

embryos

Adventitious regeneration:Why?

Organogenesis can be used for reliable clonal propagation of plants

propagation rates via organogenesis can be much higher than by axillary shoot proliferation

Many types of organs can be used as explants for shoot organogenesis

The organ of choice depends on the plant species

Floral part

Leaves

Petioles

Roots

Seeds or specific parts of seed

Stem

What type of explants to grow in morphogenesis culture medium.

Organogenesis is a multi-step process and consists of three stages including:

shoot bud formation

shoot development

rooting of shoots

The first step is the most critical part of the process since the other two are usually

consequence.

Robinia pseudoacacia (Regeneration)

Induction and formation of an adventitous bud in populus

Propagation by direct organogenesis

In certain species, adventitious shoots which arise directly from the tissues of the explant (and not within previously-formed callus) can provide a reliable method for micropropagation.

The induction of direct shoot regeneration depends on the nature of the plant organ from which the explants was derived, and is highly dependent on plant genotype.

Pea leaves with direct caulogenesis

Direct Organognesis in pea

Direct organogenesis from root pieces

In vitro shoot regeneration from root pieces is mainly reported from plants that possess thick fleshy roots such as those of the genera : Cichorium, Armoracia, Convolvulus, and Taraxacum.

It is, however, a method of propagation that is potentially applicable to a wide range of species.

Usually root explants come from isolated in vitro root cultures.

Shoot induction often occurs after the addition of a cytokinin to the medium.

• Seeliger (1956) obtained shoot buds on cultured roots of

Robinia pseudoacacia and Torrey (1958), shoot buds on root

cultures of Convolvulus.

• Direct shoot formation was induced in three species of

Nicotiana and on Solanum melongena by Zelcer et al. (1983) but

in N. tabacum and N. petunoides shoots were only obtained

after callus formed on the roots.

• The most optimistic report seen comes from Mudge et al.

(1986), who thought that the shoot formation, which they could

induce in raspberry root cultures would provide a convenient

and labour-saving method of multiplying this plant in vitro.

Direct and indirect regeneration on explants depends on explant size.

o small disks show the greatest

increase in area and fresh

weight, but the lowest

frequency of root production

o 7 mm disks of var. italica

leaves produced 10 times more

shoots per disk than 4 mm

ones.

In Brassica oleracea of vars. fruticosa and acephela

Direct and indirect regeneration frequently depends on explant size.

o Similarly, as stem internode sections of Brassica monnieri were

increased in length from 1 to 4 mm, a greater proportion of them

formed shoo t buds, and thenumber of shoots per explant increased.

o The efficiency of shoot production (i.e. the number of shoots formed

per mm of internode) was however inversely proportional to explant

length, except that those smaller than 1 mm failed to show any organ

formation

Effect of genotype on direct regeneration (1).

Plants vary considerably in their ability to produce

adventitious shoots directly on tissue explants.

Genera which produce plantlets readily from severed

leaves placed in soil or compost in a greenhouse (e.g.

Begonia, Peperomia, Saintpaulia, and Streptocarpus)

also give rise to large numbers of adventitious shoots

directly from leaf or petiole segments when these are

cultured on an appropriate medium.

Some current applications Several ornamental plants are at present propagated in vitro by direct shoot regeneration.

Chief among these are plants of the family Gesneriaceae,

(including Achimenes, Saintpaulia, Sinningia and Streptocarpus),

where shoot buds can be freely regenerated directly on leaf

explants without the formation of any intervening callus phase.

Effects of nutrients

•The requirement for both forms of nitrogen in a particular plant

species can only be determined by a carefully controlled experiment:

simply leaving out one component of a normal medium gives an

incomplete picture.

•For example, cotyledons of lettuce failed to initiate buds when

NH4NO3 was omitted from Miller (1961) salts and instead formed

masses of callus (Doerschug and Miller, 1967): was this result due to

the elimination of NH4

Nitrogen supply:

Morphogenesis is influenced by the total amount of nitrogen provided in the medium and, for most purposes, a supply of both reduced nitrogen and nitrate seems to be necessary

Effects of plant growth regulators: cytokinins

The formation of adventitious shoots, whether directly from

explanted tissues, or indirectly from callus, is regulated by an

interaction between auxins and cytokinins.

Auxin and Cytokinin interaction

• Skoog and Miller (1957) found that shoot formation could be

induced predictably from tobacco callus using relatively low levels

of auxin and a high level of cytokinin in the growth medium.

• Since this discovery, many aspects of cellular differentiation and

organogenesis in tissue and organ cultures have been found to be

controlled by an interaction between cytokinin and auxin

concentrations.

• The balance between the two sorts of regulant that is usually

required to initiate growth or differentiation in tissue cultures,.

• Relative proportions of auxins and cytokinins do not always

produce the typical results shown in the figure.

Auxin and Cytokinin interaction

Genotype is an important characteristic

The inheritance of regeneration showed that this potential have a genetic basis

Inheritance of regeneration in petunia

Petunia

7 g 14 g 21 g 28 g 34 g 41 g

0

20

40

60

80

100

L KxL LxK K

Frequency of explants with callus

a)

Frequency of differentiated explants

0

20

40

60

80

100

K KxL LxK L b)

0

1

2

3

4

5

6

7

8

9

1 0

1 1

1 2

1 3

0 0 0 0 0 07g 14 g 21 g 28 g 34 g 41 g

K

L

KxL

LxK

c)

Average number of differentiated

shoots per explant

Caulogenesis and rhizogenesis in Saintapauilia

MS salt based formula

Sucrose 30g l-1 NAA 0,5g l-1 BAP 0,5g l-1

no substrato MS

substrato MS [1/2]

substrato MS [normale]

substrato MS [2x]

Effects of salts on regeneration

no sucrose

Effects of sucrose on regeneration

no BAP

Cytokinin effects

Auxin effects