Nucellus culture

1. 1. Media preparation
2. Explant selection
3. Establishment of explant in media
4. Callus development
5. Plantlet development
6. Hardening or acclimatization
7. Open field planting
Lecture 13: Nucellus culture
Course Code : HRT 552
Course Title : BIOTECHNOLOGY OF HORTICULTURAL CROPs

3. Explant:-
Totipotent somatic cell are used.
Part of the living plant used for tissue culture
Nucellus is mass of cells enclosed within integument
Nucellus cultures are those cultures in which embryos are
derived from the nucellus or integument.
Nucellus cultures are taken from pollinated flowers and
widely reported in the genus of Citrus.
It is widely utilized to study the formation of embryos.

4. Nucellus is mass of diploid cells present in between integument and ovule

5.  The nucellus grows by additional divisions in subepidermal layers (Nikiticheva
2002).
 It surrounds the embryo sac and is five to 10 cell layers thick in the area of the
antipodal cells, with only one or a few layers nearer to the egg apparatus.
 Cells of the nucellar epidermis have a different, more compact shape, denser
cytoplasm, and, as in rice (Krishnan and Dayanandan 2003).
 There are no plasmodesmata to the integument cells (Norstog 1974).
 The nucellar epidermis persists for some time, while the mass of the nucellus
degenerates after fertilization (Domínguez et al 2001).
Nucellus is mass of diploid cells present in between integument and ovule

6. Nucellus culture help to develop virus free tissue cultured citrus plant
Nucellus is extracted after pollination but before fertilization
Nucellus is diploid (2n)
 Citrus ringspot virus
Uniform and virus free rootstock

9. Malt extract - Malt extract is sugar extracted from malted grain. This concentrated and
condensed extract is used to create wort by homebrewers.
Casein hydrolysate - Casein protein is a protein in milk that gives milk its white color. Cow's
milk is made up of around 80% casein. ... Casein peptide products might be called "casein
hydrolysate" or "hydrolyzed casein protein" on the label.
Adenine - Adenine is also used elsewhere in the cell, not just in DNA and RNA, but it's part
of the molecule adenosine triphosphate, which is the energy source for the cell. So adenine
plays a dual role in the cell: it's used for building DNA and RNA, but it's also used at storing
energy in the cell.
White’s medium - White medium is a defined medium, which consists of inorganic salts,
vitamins and carbohydrate. ... Sucrose serves as the carbohydrate source. Medium is
devoid of agar; hence this component has to be added to the medium prior to use.

10. Excise nucellar tissue
from pollinated but
unfertilized ovule
Culture in media
containing malt extract
and adenine
Embryoid is formed
Excise of embryoid and
culturing in media rich in
gibberellic acid
Disease free plantlets are
formed

11. Application of nucellus culture
1. Production of disease free plantlets
2. Very useful to get rid of viral diseases in citrus
3. In various species of Citrus nucellar monoembryony and polyembryony
occur. When nucellar tissue of polyembryonic species of Citrus excised
after pollination is cultured on White’s medium supplemented with
casein hydrolysate then callus is formed. This callus produces several
pseudobulbils, most of which form embryoids and ultimately produce
seedlings.

12. 4. Nucellar tissue excised from unfertilized ovules having no adventitive embryoids if
cultured on a medium containing malt extract and adenine then embryoids are formed
but they are unable to germinate. When such embryoids are excised and cultured on a
medium containing gibberellin, then plantlets may be formed which can be transplanted
to soil.
5. Nucellus of monoembryonic species of Citrus (such as, C. limon, C. reticulata, C.
grandis etc.) excised from post-fertilised ovules and cultured on White medium con-
taining malt extract produce few embryoids directly without callus formation.
6. From such embryoids healthy plantlets can develop within 6-7 weeks. In nucellus
cultures of Malus domestica and Vilis vinifera polyembryony have been noted. By
nucellus culture disease free clones can be obtained.

13. Disadvantages
1. Hard & complicated
2. Very precise (excise & timing)
3. Complicated process

14. THANKS 