In this article we will discuss about:- 1. Anther Culture Technique 2. Microspore Culture 3. Diploitization 4. Anther Vs. Pollen Culture 5. Plant Regeneration 6. Characteristics of Microspore Derived Plants 7. Ontogeny of Androgenesis.
The term haploid refers to those plants which contains gametophytic number of chromosomes. Anther/pollen culture mediated production of haploid plants approves the concept of totipotency. Guha and Maheshwari in 1964 reported direct development of haploid embryo from pollen of Datura innoxia by anther culture.
In an extended work, Bourgin and Nitsch in 1967 were able to accomplish complete haploid plants in Nicotiana tobacco. Since then there have been upsurge interest in the production of haploids due to their considerable potential for plant breeding.
One of the bases for haploid productions vitro is due to application in cell biology, genetics of higher plants and haploid breeding. Haploids can be exploited to detect and recover mutants in the plants. In plant mutation breeding, mutants are recessive and are difficult to recover due to heterozygous nature of plant.
However, haploid exhibit only one set of alleles and facilitates easy detection of mutants. In addition, doubling of haploid chromosomes leads to the production of homozygous plants in short span of time. The time taken for haploid production using conventional breeding requires several years. Therefore, anther culture offers a novel tool for the production of haploid.
Anther Culture Technique:
The sterilization of flower buds is initiated by sterilizing agents such as sodium hypochlorite (2%) or calcium hypochlorite (5%). After repeated rinse with sterile distilled water to remove all tracers of mercuric chloride, flower buds are carefully inscised under aseptic environment and sepals and petals are completely removed.
The anthers are then dissected out and cultured on agar medium. Anther filaments are cut off carefully to avoid damaging of anther. The damaged portion in the anther may tend to develop callus. Generally 6-10 anthers are placed in the culture vessel. Observation in tobacco anther culture shows that anthers cultured on media turn brownish within 12-15 days (2 weeks).
After 2-3 weeks in culture, small callus like protruberance is seen and entire callus mass is emerged or burst out from the anther or embryo emerged out and develops into plantlet. The callus may also differentiate to plantlets. The plantlets emerged out of the anthers in 4-5 weeks (Fig. 9.1).
In the case of microspore or pollen culture, anthers are excised and about 10-15 numbers (anthers) are transferred into sterilized beaker. The anthers are then crushed against wall of the beaker using glass rod. Pollens are squeezed out to collect at the bottom of the beaker containing suitable aqueous media. The anther extract are selected for centrifugation (100-150 g) and collected the pellet of pollen grains. The isolated pollen is washed and subjected to culture.
Microspore Culture:
Certain culture method like nurse culture is generally followed by isolated microspore culture. In this method, anther of mother plant is placed horizontally in the container supplied with shallow layer of liquid media. A filter paper disc is placed above the anther and about ten pollen grains are placed on the filter paper disc. The proliferation of microspore results in cluster of parenchymatous cells within 1 to 2 weeks. These clones were then transferred to agar culture media for successful induction of androgenesis.
Diploitization:
Haploid plants can produce flower, but they are sterile. In order to ensure the production of fertile haploid plants, diploitization process is required. Diploitization is accomplished by treating plantlet obtained by anthers directly in colchicine solution for 24 hrs.
In another method colchicine paste can be prepared and applied directly on the axils of in vitro haploid plantlets. After colchicine treatment, homozygous diploid plants are obtained due to the duplication of chromosomes. These homozygous diploid plants are fertile.
Anther Vs. Pollen Culture:
Although anther is generally used to obtain haploids, its microspore in anther is surrounded by several diploid tissues like connective tissues, middle layer, and tapetum. Any damage or growth stimulation in vitro may tend to the proliferation of diploid tissue and consequently responded culture will be mixed with haploid and diploid cells, which is difficult to separate in vitro.
The problem has been evidenced in several cases. Anther culture in Helianthus annus exhibit high reactivity of somatic tissues in anther such as anther wall, multicellular hair type structure, anther connective and parenchymatous vascular buds. Microspore culture was invariably assayed in order to avoid the reactivity of somatic tissues.
Microspore culture often provides unique tool to overcome the proliferation of anther somatic tissue. Microspore derived plants ensures complex nature of haploids in vitro. Some of the drawbacks of microspore or pollen culture is that the lack of essential nutrients normally provided by tapetal cells and also density of the pollen grain.
Plant Regeneration:
Regeneration from embryo is a simple procedure and can take place on the induction medium. While most of the media containing growth regulators and high sucrose levels, plantlets development fail to occur unless the embryos are shifted to embryo culture media, which is free from hormones and low sucrose concentration. When callus is produced from microspore, regeneration is accomplished on hormones free or low level of auxin with increased level of cytokinin.
When auxin is essential, certain auxins like IAA is often employed. Benzyl adenine and kinetins are the commonly used cytokinin in the medium. In cereals, regeneration frequencies occur at less frequency and still decreases with increasing callus age. Appearance of albinos due to the 2, 4-D, is another serious problems in regeneration of cereals.
Characteristics of Microspore Derived Plants:
The majority of microspore derived plants are haploids, but in some cases, both diploid and polyploids may be present as in case of tobacco. Aneuploids and mixaploid chimeras have also been sighted commonly in vitro. Several occasions, evidence for homozygous natures of anther derived diploids are seen from genetic is biochemical analysis. Convincing evidence based on cytological studies reveals that diploids and polyploids problem arise due to the nuclear fusion or endureduplication in microspore during culture. In addition, culture temperature is also added to the problem in vitro.
Ontogeny of Androgenesis:
Anther culture shows various modes of microspore development leading to androgenesis. They are direct and indirect androgenesis.
Direct Androgenesis:
During microspore development, various stages of embryogenesis are followed due to the behaviour of microspore like zygote. The embryo is released at globular stage from exine and continues to develop further and finally cotyledons are transformed into plantlets.
Indirect Androgenesis:
In the process of indirect androgenesis, microspore undergoes multiple divisions to a mass of callus, which burst through anther wall. Induction of callus is manipulated by composition of media. Regeneration from the callus can be accomplished by adjusting hormones concentration. The callus is differentiated to form embryo or shoots and further transformed into plantlets.