(A) Anther Culture:
Though in vitro culture of anthers was first undertaken by Shimakura (1934), it were Guha & Maheshwari (1964) of Delhi University who first successfully obtained numerous embryolike structures called embryoids and subsequently the plantlets from mature anthers of Datura innoxia.
These two workers observed the embryoids developed from the inside of the anthers at that time but in 1966 they confirmed that the origin of embryoids has been the regeneration of plants from anthers (androgenesis) as well as from excised pollen grains has been of much interest.
The intact anthers containing the developing pollen have been cultured in numerous laboratories of the world and this has resulted in the development of embryoids directly from pollen grains. These embryoids have been induced to develop into whole plants which are true haploids (with reference to higher plants, haploids represents the gametic chromosomes constitution). (N.B. seeds of most flowering plants are diploids). It is evident from a recent review that most species capable of haploid production proceed via anther culture or androgenesis.
There is a long list of species in which haploids have been produced by anther culture technique. Some of these are Datura metal, Nicotiana tabacum, N. sylvestris, N. alata, N. rustica, N. glutinosa, Solanum nigrum, Lycopersicon esculentum, Petunia hybrida and Capsicum annum.
As it is apparent, the solanaceous plants respond well to another culture technique probably because of the fact that they have long-sized anthers and the latter flourish well in almost every culture medium. However, the following are crop species capable of haploid plant production from another culture: rapseed, tobacco, rice, rye, potato, corn, asparagus, pepper, strawberry, rubber, barley, sweet potato, wheat, grapes and clover.
For successful anther and excised pollen culture, it is essential to excise the flower buds at the correct time the correct time is the stage of the first mitotic division of the uninucleate microspores tetrad. Buds are surface sterilized with alcohol, chlorine-water, sodium hypochloride or ethyl mercuric chloride. Now, the anthers are excised and transferred onto nutrient medium.
Different nutrient media used for anther culture are Murashige-Scoog media, Nitsch medium and certain other modifications -1% agar is added to the medium to provide solid base. The transferred anthers, in the beginning, show slight swelling followed by initiation of cell division inside the massive connective tissue.
These latter show initiations of light green crumbled callus. Simultaneously, the pollen sac swells up and raptures along its upper margin lengthwise exhibiting a mass of pale-green pollen-callus. The callus arising from pollen shows slow growth as compared to connective callus.
Now, the nutrient medium is supplemented with high concentration of kinetin for multiplication of callus, keeping the auxin concentration quite low. However, when formation of root and shoot is desired, the ratio of kinetin and auxin concentration is reserved in the medium.
(i) Contrary to the fact that mutations are difficult to detect in higher plant because they are usually recessive and do not express themselves in the presence of their dominant alleles on the homologous chromosomes, the mutations induced in haploids can be easily detected because they have only a single set of genes and, consequently there is no interference by their dominant alleles.
(ii) Haploids provide a source of haploid tissue. Suspension of haploid cells can be obtained from haploid tissue. These haploid cells can be used to carry out new genetic researches on higher plants by applying the techniques used for microorganisms.
(iii) Haploids can be easily treated with colchicine to obtain homozygous diploids in a single generation. It is much advantageous to the traditional plant breeding methods because it involves selling for many generations in many years to obtain homozygous diploids when conventional method of plant breeding is applied.
(B) Ovary Culture:
(a) Technique LaRue (1942) was probably the first who successfully obtained in vitro rooting of the pedicel and a limited growth of ovaries. Nitsch (1951) developed this technique by growing excised ovaries of some plants (e.g. Cucumis angaria, Nicotiana tabacum, Lycopersicon esculentum, Phaseolus vulgaris) in vitro under aspetic conditions, the excised ovaries from pollinated flowers of Cucumis anguria and Lycopersicon esculentum viable seeds.
But, however, the fruits so developed were comparatively smaller than those naturally developed. Maheshwari and Lai (1958) excised the ovaries from flowers of Iberris amara one day after pollination, grew them on a simple midium containing mineral salts and sugar supplemented with B-Vitamins and obtained normal healthy fruits matching the naturally developed ones.
(i) Fruits developments:
The knowledge of fruit physiology is valuable in improving the quality of fruits. The technique of excised ovary culture may allow one to study the effect of chemicals directly on ovaries. Contrary to it, when the entire plant is employed with chemicals to study their effects on ovaries, it remains questionable whether the chemical used act in its original form or its originality is somewhat altered in the plant system before it reaches the ovary.
(ii) Parthenogenetic Haploid Production:
Hess and Wagner ((1974) successfully obtained parthenogenetic haploids of Mimulus luteus cv. tigrirus grandiflorus by pollinating their exposed ovaries in vitro ovary culture studies. Similar results of producing parthenogenetic haploids have been obtained in the cultures of unfertilized ovaries of Hordeum vulgare, Nicotiana tabacum and Triticum aestivum.
Nucellar tissue excised from post pollinated carpels, when the adventive embryos (the embryos arising from the maternal sporophytic tissue, outside the embryosac, are called adventive embryos), are already in torpedo-shaped stage, and cultured on case in hydrolysate supplemented with White’s medium, proliferate and result in the development of callus. Numerous outgrowths, referred to as pseudobulbils, differentiate from the callus.
The pseudobulbils develop into embryoids which germinate and finally, give rise to seedlings. It has been found for the first-time by Button and Bormann (1971) that the excised nucellar tissue from unfertilized ovule (i.e. the case where there are no adventive embryos present), when grown in vitro under suitable conditions, develop embryoids. When the embryoids are excised and planted individually on a medium supplemented with GA,, they sometime form plantlets. These plantlets have been successfully transplanted to soil by Button and Borman.
The nucellar tissue culture may prove of great significance in horticulture. The seedlings obtained from embryoids are of parental type, as obtained through vegetative propagation by cuttings. However, nuclear seedlings of Citrus furnish better clones of orchard stock than cuttings as a result of the following reasons:
(i) The nucellar seedlings, have a tap root, and, therefore develop a better root system than do the cuttings. The latter have only a small lateral root system.
(ii) The nucellar seedlings generally restore the vigour which is lost after repeated propagation by cuttings.
(iii) The nucellar embryos prove to be disease free. The nucellar polyembryony has been used as a significant practical approach to develop virus-free clones of polyembryonate Citrus varieties.