Factors Affecting In Vitro Androgenesis | Genetics

The following points highlight the nine main factors affecting in vitro androgenesis. The factors are: 1. Age and Physiology 2. Stages of Pollen 3. Thermal Stress 4. Genotype 5. Chemical Treatment 6. Medium Factor 7. Anther Wall Factor 8. Microspore Density 9. Cultural Conditions.

Factor # 1. Age and Physiology:

The physiological conditions of the donor plant have a considerable effect on the response of microspore in culture. Plant age is a significant factor in determining high frequency of embryogenesis. Thus flower buds are collected from the plant in the beginning of flowering season rather than at the end of flowering season.

Generally, plants grown under green-house conditions ensure disease free and physiologically active status of the plant from which buds are collected for anther culture.

Factor # 2. Stages of Pollen:

The stage of pollen grain in anthers plays a crucial role in determining androgenesis. Thus, immature anthers containing uninucleate pollen at the time of first mitosis are the ideal stage for the induction of androgenesis.

Even just before or after first pollen mitosis are also ideal stages in anther for selection. One of the possible reason choosing particular stages of pollen is that tRNA genes are active and turned on at these stages.

Several other physiological factors could influence androgenesis in culture. The plants which are grown under low temperature generates greatest embryo yield. Experiments with tobacco shows the variations in photoperiod and light intensity influence microspore embryogenesis with higher yields of embryo observed when donor plants are grown under short days and high light intensities.

Even seasonal variation is important while selecting flower buds. The callus produced from barley exhibit higher frequency when selected in later summer, whereas in rape seed, highest embryo is obtained in spring and early summer. In certain cases, field grown plants yield better results than green-house grown plants.

Factor # 3. Thermal Stress:

Pre-conditioning of the anthers prior to culturing is highly desirable, which is receiving increasing attention recently. Low temperature, pre-treatment of anthers for periods of 2-30 days at temperature, from 3 to 10°C are known to stimulate embryogenesis. Other treatment includes maintenance of detached inflorescence in water for several days.

It was shown that flower buds subjected for cold and hot treatment or combination of both are useful in producing androgenesis. Exposure of plants to thermal shocks can change the pattern of division of microspore nucleus. In wheat, before removal of anthers, spikes were subjected to temperature stress of 3°C.

Factor # 4. Genotype:

Induction of androgenesis is more successfully governed by plant genotypes. Various species and cultivars exhibit different response in culture. This has been noticed in the cultiva­tion of Arabidopsis thaliana and Lycopersicum esulentum in which only three cultivars are able to produce haploids. Similar observations are seen in anther culture.

The better situation was noticed remarkably in various species in Nicotiana and solanum. These studies clearly indicate that these experiments should be carried out with all the remaining cultivars. Androgenesis is heritable, and ability to undergo callus formation is controlled by few nuclear genes. The possible beneficial effect of cold treatment (2°-5°C) is induction of androgenesis due to the acceleration of pollen viability and delay in senescence.

The genetic factors affect greatly in the androgenic ability of Triticum. The close relation of wheat, spelta, containing q-factor located on chromosome 5A is probably responsible for its adaptability to grow in wide range of climatic conditions. Thus, anthers taken from Triticum spelta, a hybrid between wheat or spelta exhibit a great potential haploid production.

Factor # 5. Chemical Treatment:

Plants treated with several chemicals such as malic hydride, colchicine increases effi­ciency of haploid induction in Beta vulgaris and Nicotiana langsdorfi. In addition, spraying growth regulators or hormones also provided considerable results.

Following the applications of 2-chloro ethyl phosphoric acid, commercially known as ethrel, causes male sterility. Forma­tion of multinucleate pollen due to additional mitosis occurs in wheat following ethrel spray before first meiosis in the pollen mother cells.

Factor # 6. Medium Factor:

Success in androgenesis is controlled well by the composition of medium. Several media formulations have been designed to accomplish in vitro response, that includes several inorganic nutrients, nitrogen in particular and organic supplements like amino acids. Media formulations designed by Nitsch (1969), basal media of MS encouraged androgenic development in wide range of species.

Sucrose plays a prominent role in embryogenesis from anther or microspore culture. Addition of 6% is required for androgenesis in most cereals. Microspore viability has been noticed during the early stages of anther culture in presence of 15-16% sucrose as in the case of Brassica napus.

Production of haploids were noticed in neem anther culture by supplying 9% sucrose. Anther culture of sunflower preferred 10% sucrose. It however, shows that embryo germination was largely increased by decreasing sucrose concentration to 10% > 6.1 > 3%.

Similarly, Chen and Dribnenk (2002) employed NLS media with 12% sucrose for the haploid production in flax. Sucrose is supplied as chief carbohydrate source in culture media. The concentration required by tobacco microspore is less (4%) whereas its requirements for wheat and rice at elevated levels range from 6 to 12%.

The requirement for sucrose is very specific and none of the other disaccharide other than glucose to some extent can substitute sucrose. Several amino acid supplements like glutamic and serine stimulates embryogenesis in tobacco and rape seed. The culture medium enriched with inorganic nitrogen promotes androgenesis in rice anther culture.

Other beneficial agents include coconut milk, ascorbic acid, nucleic acid hydrolysate are employed in culture media. Due to the undefined nature of coconut milk it can be replaced by yeast extract. The modified Chinese media gives the best result for embyro induction and regeneration.

Growth regulators play a role in induction of androgenesis. Auxins such as 2, 4-D in the media are essential for callus formulations in barley, rice and wheat. Studies with cereals indicate the auxin usage can be avoided if media formulations are designed specifically. Utility of auxin could be seen during initial stages of culture and could be subsequently recovered.

Cytokinins are indispensable on several occasions and have been approached in the anther culture of Datura and potato. Gibberellin exhibits some encouraging results in case of Datura. However, certain plants particularly in microspore culture of B. napus have revealed that exogenous supplies of growth regulators are not essential and many are inhibitory to anther response.

Utilization of liquid media in anther culture has clear advantages over solid media to increase frequency of embryogenesis in several plants and has been evidenced in tobacco and barley culture. The negative role of solid media on several occasions is probably due to the accumulation of inhibitors like impurities in agar-agar.

Enhanced yield of embryo and callus obtained in culture by the addition of activated charcoal which is probably involved in the removal of inhibitors by adsorption process. Generally, two percent charcoal is used in the medium enhancing embryogenic frequency. In sunflower (Helianthus annus) androgenesis in vitro have been improvised by the addition of 0.1% polyvinylpyrrolidin.

Increase in the yield of androgenic anthers from 15 to 40% was observed in tobacco. In the enhanced frequency of androgenesis in tobacco certain chemicals have been implicated due to the absorption of hydroxymethyl furfural.

Factor # 7. Anther Wall Factor:

Induction of tobacco androgenesis can occur in media containing only agar and water or on simple media formulations indicates that the anther wall may play a critical role during early stages of culture. Tapetal cells, which outlines the anther wall provides essential nutrition to the development of pollen mother cells into mature pollen.

Therefore, in the entire anther culture system the presence of anther wall provides essential nutrients. The existences of inhibition wall factor have also been implicated.

Factor # 8. Microspore Density:

In microspore culture for example, only a fraction of total microspore population will undergo division. Microspore density may be a crucial factor determining culture success. The isolated microspore density of 105 microspore/mL has been determined to play a prominent role in the successful culture.

Factor # 9. Cultural Conditions (Light and Temperature):

Light and temperature decides nature of tissue proliferation but many contradictions have been reported: Continuous light supply during the culture in initial stage is detrimental to tobacco anther culture. Interesting results were noticed during photoperiodic treatment, are believed to be beneficial.

Red light is stimulatory to embryogenesis in isolated microspore. In addition, incandescent and fluorescent light greatly enhances embryo production in tobacco. High light intensity (3-200 lux) is beneficial to rice callus formation and inhibitory to Nicotiana microspore but low intensity of 300 lux in beneficial.

Culture temperature has a significant role in microspore embryogenesis. For example, Datura embryo fail to form unless temperature is maintained at 20°C and its responses increased upto 30°C. In certain cases preconditioning of anthers at elevated temperature may stimulate androgenesis. Anthers of Brassica campestris maintained at 35°C for 24 hrs before transferred to 25°C greatly encouraged androgenesis.