Culture of Flower, Ovary, Ovule, Seed and Nucellus | Plant Tissue Culture

In this article we will discuss about the Culture of Different Parts of Plants:- 1. Culture of Flower 2. Culture of Ovary 3. Culture of Ovule 4. Culture of Seed 5. Culture of Nucellus.

Culture of Flower:

Angiosperm flowers were first cultured by Larue (’42). He noted some growth of ovaries and rooting. Complete flowers of several dicots were successfully cultured by Nitsch (’51). Flower primordia of Rananculus scleratus were cultured by Konar and Nataraj (’64, ’65). They obtained plantlets from such cultures.

Medium:

Konar and Nataraj used White’s medium containing sugar and ferric salt for culturing flower primordia of R. scleratus. On such culture floral primor­dia produce callus, from which many embryoids are formed. These embryoids on sub­culture produce plantlets.

In cucumber (Cucumis sativus Linn.) when young male flower buds were cultured on a medium containing inorganic salts, vitamin B, casein hydrolysate, coconut milk, then these buds tend to form ovaries. This tendency is increased by the addition of IAA to the medium.

Method:

Nitsch (’51) selected flowers two days after pollination and surface sterilised the flower with 5% hypochlorite solution. These were then thoroughly washed with double distilled water and placed on culture medium containing inorganic salts and sucrose.

On culture such flowers produce fruits. Larger fruits were obtained on medium supplemented with hormones, such as, IAA, gibberellin, cytokinin. Flowers excised before pollination cannot produce fruits.

Culture of Ovary:

Horticulturist tried to improve the quality of the fruit by using various chemicals. In such studies, the culture of excised ovary supplied directly with the chemicals, has great importance.

Nitsch (’51) successfully cultured excised embryos of several plants, such as, Lycopersicon esculentum (Fig. 12), Nicotiana tabacum, Phaseolus vulgaris, Cucumis anguria, Frageria sp. etc. The excised ovaries of Cucumis and Lycopersicon produced fruits with viable seeds on culture. But these fruits are smaller than normal.

Maheswari (’58) successfully cultured ovaries of Iberis amara excised one day after pollination on a medium containing mineral salts and sugar. In such a medium smaller embryos are formed. But if the medium is supplemented with B-vitamins then healthy fruits of normal size develop.

Addition of IAA to this vitamin contain­ing medium further improves the size of the fruits. Ovaries excised at zygote stage develop normally in culture as seen in Hyoscyamus niger (Bajaj ’66) and Anethum graveolens. If coconut milk is added to the medium then larger fruits are produced.

In several plants, such as, Allium, Althea, Hordeum, Hyoscyamus, Iberis, Triticum etc. it has been noted that the presence of sepals helps fruit development. In absence of sepals fruit development is poor. Guha and Johri (’66) demonstrated this in Allium.

Nitsch’s medium supplemented with vitamin, glycine and yeast extract is most suitable for ovary culture. Ovaries of Linaria moroccana were grown on this medium. The fruits produced on this medium were usually smaller than the natural size. In Tropaeolum also the fruits produced on culture were smaller.

Ovaries excised after pollination can produce fruits on simple medium containing mineral salts, sugar and, vitamin. Ovaries taken from un-pollinated flowers fail to produce fruits on such a simple medium. Un-pollinated flower may develop into seedless fruits on a medium supplemented with hormones.

In Lycopersicon Nitsch (’51) obtained seedless fruits by culturing un-pollinated ovaries on a medium supplemented with 2-4-D or 2-naphthoxyacetic acid. Chopra (’58) obtained parthenocarpic fruits of Althea rosea on a medium containing indole butyric acid.

Pollination stimulus is required for ovary and seed development even by most apomicts. Only female plants of Aerva tomentosa growing in Delhi is an apomict. Puri (’63) cultured excised spikes, flowers, ovaries, etc. of this plant and noted that on a medium supplemented with IAA and yeast extract portions of spike grow well and produce seeds, which are comparable to that growing in nature.

Fruits produced on ovary culture usually contain few seeds or poorly developed seeds or no seeds. Nitsch (’51) observed this in ovary culture of Lycopersicon and Ni­cotiana. But normal seed development was noted on ovary culture of Iberis, Hyoscy­amus and Linaria on basal medium. Anethum requires a supply of coconut milk for normal seed development.

Significance:

(1) By ovary culture the physiology of fruit development can be studied.

(2) By ovary culture haploids can be produced. Unfertilised ovaries of Hordeum vulgare, Nicotiana tabacum and Triticum aestivum develop into haploids.

Parthenogenesis haploids of Mimulus luteus cv. tigrinus grandifiorus were raised by Hess and Wagner (’75) by pollinating the exposed ovules in ovary culture.

(3) Dormancy period of seeds can be reduced by ovary culture. Achenes of Ran­unculus sceleratus remain dormant for one year under normal conditions. But the ach­enes of this plant produced on ovary culture have no dormancy period.

(4) Rare hybrids can also be produced on ovary culture.

Culture of Ovule:

White in 1932 first attempted to culture the ovules of Antirrhinum majus. The method of ovule culture was improved by Maheswari (’58, ’61). He cultured ovules of Papaver somniferum excised 6 days after pollination in Nitsch’s medium and obtained viable seeds. Initially the embryos grew slowly but after globular stage the growth was rapid.

Like Papavar, ovaries of orchids excised after pollination grew normally on a simple culture medium containing 10% sugar. Culture of ovules excised at or after globular stage produced mature seeds easily.

This has been noted in several plants, such as, Allium cepa, Gynandropsis, Impatiensand Nicotiana tabacumetc. Ovules excised soon after fertilization, before reaching globular stage, usually failed to produce mature seeds on culture. Such ovules require a special medium for proper growth.

Ovules from various plants excised at early stages, such as, zygote stage or 2- celled proembryo stage, show diverse growth requirements on culture. Thus, ovules of Zephyranthus required a medium supplemented with coconut milk and casamino acid for normal embryo development and formation of viable seeds.

In Trifolium repens for proper growth of the ovule a medium containing fruit-juice of cucumber or watermelon was required. In Petunia hybrida ovules excised 2 days after pollination grew on a medium supplemented with fruit juice of cucumber.

It has been noted that the presence of pla­cental tissue favours development of ovule in culture. This was observed in ovule culture of Gynandropsis by Chopra and Sabharwal (’63).

When excised ovules of Gossypium hirsutum was cultured on a medium supplemented with casein hydrolysate or its ovule extract then tannin accumulated on cotton fibre initials and the fibre initials collapsed. But when the ovule excised two days after anthesis was cultured on a medium containing gibberellin at 30°C then good fibre development was noted.

In some interspecific crosses hybrid seedlings failed to develop even by embryo culture technique. By culturing ovules 3 days after pollination of Gossypium arboreum X G. hirsutum on Murashige and Skoog’s medium Pundir (’67) was able to produce hybrid seedlings. Using similar method Steward and Hsu (’78) produced seedlings of different interspecific hybrids of Gossypium.

Significance:

(1) Zygote or very young embryos which cannot be cultured easily can be grown by ovule culture.

(2) Developmental stages of zygote or young embryo can be studied in ovule culture.

(3) Nutritional requirements of young embryos can be studied.

(4) In some interspecific crosses where embryo culture fails to raise the seedlings, ovule culture may be the effective method to raise viable seedlings.

Culture of Seed:

Some plants produce unorganised embryos, which are not differentiated into radicle, plumule and cotyledons. This is observed in some species of Orchidaceae and Orobanchaceae. In these plants seedlings are formed by the globose embryo directly. In Eranthis sp. belonging to Ranunculaceae the seeds contains unorganised embryos during shedding.

After the seeds have fallen to the ground, these by intraseminal growth form typical dicot embryos. In orchids during seed germination the plumular pole, that is the embryonal pole distal to the micropyle, forms a protocorm like structure, which later differentiates into roots and shoots.

Such type of seedling formation only from one end is known as ‘monopolar seedling formation’. In Orobanchaceae also monopolar seedling formation has been noted. In Orobanche crenata the radicular pole (i.e. the pole proximal to the mi­cropyle) produces the seedling.

In seed culture of Orobanche aegyptica both monopolar and bipolar seedling for­mation occurs depending upon the composition of the culture media (Usha ’68). In a medium containing coconut milk or yeast extract monopolar seedling formation occurs. If the medium is supplemented with IAA or gibberellin or kinetin bipolar seedling is formed.

In seed culture of some obligate root parasites such as Orobanche, Striga, the host stimulus can be replaced by some chemicals. The seeds cultured on a medium supple­mented with gibberellin, cytokinin or strigol can produce seedlings in absence of host stimulus.

Seed cultures of parasitic angiosperms in absence of host usually do not produce haustoria. But certain root-parasitic species of Scrophulariaceae on culture produce haustoria in absence of host when some chemically undefined substance, such as aqueous extract of gum tragacanth is present in the medium.

Some stem parasitic species of Loranthaceae (Scurrula pulverulenta) when cultured on White’s medium supplemented with ‘casein hydrolysate can produce haustoria. Rao (’63) successfully cultured seeds of some interspecific hybrids of Vanda and obtained seedlings either directly or callus is formed from which seedling later differ­entiates.

Culture of Nucellus:

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.

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.

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 con­taining malt extract produce few embryoids directly without callus formation.

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.