In this article we will discuss about the process of androgenesis, explained with the help of suitable diagrams.
A. In Vitro Pathways of Androgenesis:
The anther or pollen normally starts undergoing in the pathway of androgenesis within 2 weeks and it takes about 5-8 weeks to obtain complete plantlets.
Haploid plants are formed in two ways:
(i) Direct Androgenesis:
Embryos are directly formed from the pollen/microspore without callus.
(ii) Indirect Androgenesis:
Microspores undergoes division in unorganised fashion to give rise to callus and by embryogenic or organogenic induction the haploid plantlets may be obtained.
There are four different pathways to form the multicellular condition of pollen from the unicellular pollen (Fig. 21.4).
Pathway I:
The microspore divides by an equal division and two identical daughter cells contribute equally to the sporophyte development e.g., Datura innoxia.
Pathway II:
The uninucleate microspores divide unequally forming vegetative and generative cell. The sporophyte arises through divisions of vegetative cell, the generative cell gets degenerated e.g., Nicotiana tabacum.
Pathway III:
The uninucleate microspore undergoes a normal unequal division but the pollen embryos are formed from the generative cell alone. The vegetative cell does not divide, e.g., Hyocyamus niger.
Pathway IV:
The division of microspore is asymmetrical as in pathway II, but both the cells take part in embryo formation and sporophyte development e.g., Datura metal.
B. Factor Affecting Androgenesis:
(a) Stage of Pollen:
Anthers with microspores ranging from tetrad to the bi-nucleate stage are responsive to culture. As soon as the starch deposition starts within the microspore there is no further development towards sporophyte.
(b) Physiological Status of Donor Plant:
The anther should be collected from the flower buds of adult healthy plant and it is very important to use the healthy explant. The variation in response of anthers from plants grown under different environmental conditions may be due to the differences in endogenous level of growth regulators. Flowers from relatively younger plants, flowering at the beginning of flower-season are more responsive.
(c) Genotype of Plants:
Success of anther culture is highly dependent on the genotype of the plant. It has been observed that various species and cultivars exhibit different growth responses in culture.
(d) Pretreatment of Anthers:
The pathways towards androgenesis require to stop the development of pollen cell towards gamete formation and to force to develop the multicellular condition.
This induction can be given by different types of pre-treatments:
(i) Cold Shock:
In general cold shock or cold treatment between 3-6°C for 3-15 days may be applied. Cold treatment helps the weak and non-viable anthers/microspore to be killed, as a result the good material get more enrichment.
(ii) Hot Treatment:
In some species the hot water treatment (3’0-40°C for 24 hr-1 hr) helps more embryo development from the pollen. The temperature shock helps in dissolution of microtubules and causes abnormal division of microspores.
(iii) Chemical Treatment:
Chemicals like 2-chloroethyl phosphonic acid have a pronounced effect in increasing the haploid production in various species. It helps to develop the multinucleate condition with fewer starch grains.
(e) Culture Media:
Composition of medium is one of the most important factors determining the success of anther culture. But it is difficult to draw a conclusion about the suitable media composition for different genes/species or even genotypes.
Higher concentration of sucrose is essential for osmoticum and also androgenesis, chelated iron has been shown to induce embryogenesis. Minerals and growth regulators play important roles on embryogenesis but it totally depends on the endogenous level of hormones.
(f) Culture Conditions:
The physical environmental conditions in which the cultures to be placed can enhance the differentiation. The cultures are incubated generally at 24-28°C.
In the initial stages of induction of morphogenesis, darkness is normally more effective or cultures should be kept at low light intensity (500 lux). After formation of macroscopic structures, these can be transferred to a regeneration medium and kept at 14 hr. day light condition at 2000-4000 lux.
C. Diploidisation of Haploids:
The ploidy level of plants derived from anther or microspore culture is highly variable. This variation may be due to endomitosis or fusion of various nuclei during the developmental stages of anthers at the time of excision and culture.
For obtaining homozygous diploid lines, the plants derived through anther culture must be analysed for their ploidy level and then the following methods can be applied:
Colchicine Treatment:
As colchicine is a spindle inhibitor it is used to induce chromosome duplication in various ways:
(a) The regenerated plantlets can be treated with 0.5% colchicine solution.
(b) Repeated colchicine treatment to apical or axillary bud by cotton soaked in colchicine or by applying colchicine-lanolin paste.
(c) In case of cereals, the tillers can be put into colchicine solution cutting the crown and then again put back to soil.
(d) Anthers can be plated directly in colchicine supplemented media for a period and just after first division again the explants should be placed on colchicine-free media.
Endomitosis:
Haploids are in general unstable in culture and have a tendency to undergo endomitosis to form diploid cells. This property can be exploited in some cases to obtain the homozygous plant.