- The process of development of haploid plants from microspores or immature pollen is called as Androgenesis.
- Plant that have gametic number of chromosomes in body cells i.e. half the number of chromosomes in the somatic cells of their parents, are called as haploid plants.
- Haploid plants can develop spontaneously in nature, like Datura sp, ~ 100 species.
- Sexually sterile and cannot reproduce, but chromosome doubling can make these plants fertile and are called double haploids.
- Double haploids are homozygous, hence very important for genetic studies.
- Guha & Maheshwari, reported production of haploids in vitro for the first time in 1964.
Techniques of Androgenesis
- Anther Culture
- Pollen or Microspore Culture
Anther Culture
- Anthers are the pollen containing units of the stamens.
- Anthers at the flower bud stage are usually most responsive and cultured in aseptic conditions.
- The selected buds are surface sterilized with a suitable disinfectant.
- Anthers along with their filaments are excised under aseptic conditions and placed on a sterilized petri-plate.
- One of the anthers is crushed in acetocarmine to test the stage of pollen development and if it is found to be of the correct stage the anthers of the remaining stamens are gently detached from their filaments, without injuring the anthers, and placed horizontally on the medium (anther culture).
- When dealing with plants having minute flowers, such as Asparagus, Brassica and Trifolium, it may be necessary to use a stereoscopic microscope for dissecting the anthers.
- Alternatively, only the perianth may be removed and the rest of the bud, with the stamens intact, inoculated.
- The gap between bud collection and anther/pollen culture should not exceed 2h.
- The anther cultures are generally maintained in alternating periods of light (12-18 h; 5000-10 000 lx m 2) at 28~ and darkness (12-6 h) at 22~.
- However, optimal storage conditions need to be determined for individual systems.
- For example, the anther cultures of Brassica species are very sensitive to light and, therefore, should be maintained in the dark throughout.
Anther Culture
- Development of androgenic pollen may occur through two pathways.
- Direct Androgenesis: Pollen directly develops into embryos.
- It mimic zygotic embryogenesis.
- Indirect Androgenesis :
- After a few cell divisions, the multicellular tissue dedifferentiate and proliferate to form callus.
- Later, normal in vitro regeneration protocols are followed.
Microspore or Pollen Culture
- There are many problems associated with raising haploids through anther culture.
- The pollen grains within an anther lobe being genetically heterogeneous, the plants arising from an anther would constitute a heterogeneous population.
- Mixing of calli of different pollen origin within an anther lobe enhances the chances of regenerating chimeric plants.
- Furthermore, if the proliferation of anther wall cells occurs concomitant with the callusing of pollen the tissue finally derived would not be purely of gametophytic origin.
- In the cultures of anthers showing asynchronous pollen development the older grains may suppress the androgenic response of younger grains by releasing toxic substances as observed in Brassica napus.
Microspore culture
- Isolated pollen culture cannot only circumvent these problems but also offers many other advantages:
- (a)it is a haploid, single cell system,
- (b)a homogeneous population of pollen grains at the developmental stage most suitable for androgenesis can be obtained by gradient centrifugation,
- (c)Isolated microspores, can be genetically modified by exposing them to mutagenic treatments or insertion of foreign genes before culture and the new genotypes selected at an early stage, and
- (d)pollen culture is 60 times more efficient than anther culture in terms of embryo production.
- The initial success with isolated pollen culture was based on the use of some kind of a nurse tissue or its extract.
- Sharp et al. (1972) raised haploid tissue clones from isolated pollen grains of tomato by plating them on small filter paper pieces placed over cultured anthers of the same species.
- The pollen grains are released from the cultured anthers either mechanically or by float-culture method (cold treatment of anthers, which liberate the pollen grains after 2-7 days into the medium).
Factor Affecting in vitro Androgenesis
- Physiological Status of the donor plant
- Pollen development stage
- Anther wall factors
- Genetic potential
- Pretreatments
- Culture medium
Physiological Status of the donor plant
- Physiology of the donor plants, which is affected by its age and the environmental conditions under which it has been grown, significantly influences the androgenic response.
- Generally, the buds from the first flush of flowers show better response than those borne subsequently.
- The anthers excised towards the end of the flowering season exhibit a delayed response, in addition to a considerably low frequency of sporophyte formation.
- In Brassica rapa, however, pollen grains from old, sickly looking plants produced more embryos than those from young and healthy plants.
- Exposure of the donor plants to stresses, such as nutrient stress and water stress are also reported to promote androgenesis.
- Treating the donor plants with the substances which interfere with the normal development of pollen grains, such as etherel, auxin, gibberellin also promote androgenesis.
- In B. napus, growing the donor plants under comparatively lower temperatures improved the yield of pollen embryos.
Pollen Developmental Stage
- Selection of appropriate age of pollen grains is very critical in the induction of androgenesis.
- Generally, the pollen grains around the first mitosis are most responsive.
- The early bicellular stage of pollen is best for Atropa belladonna and Nicotiana sylvestris and absolutely necessary for N. knightiana.
- Kott et al. (1988) have reported that in B. napus binucleate pollen grains are not only non-embryogenic but can also suppress embryo formation by the embryogenic, uninucleate grains by secreting hydrophilic, heat stable toxins.
- The stage of pollen development at culture may also affect the ploidy level of the pollen plants.
- In anther cultures of Datura innoxia and Petunia sp., while uninucleate microspores produced mainly haploids the binucleate pollen formed plants of higher ploidy.
Genetic potential
- Donor plant genotype influence its androgenic potential significantly. Even the genotypes of same species show considerable variations. like androgenic response of japonica cultivars rice is much better than Indica cultivars.
- To improve androgenic response, crossing of cultivars having poor responses with the cultivars with higher androgenic potential, is useful.
Media Composition
- Media usually contain low salt concentration as compared to MS medium.
- Cereal pollen is sensitive to inorganic nitrogen source like ammonia.
- Sucrose (2-4%) is commonly used carbon source, however its concentration may vary. 6% in wheat and ~12% in Brassica sp.
- Maltose is more successful in cereals.
- Plant hormones are required at low concentration.
- Agar may contain gelling agents that are inhibitory to androgenic process.
- Liquid media can also be used for anther culture.
Pretreatments
- Application of stress promotes induction of androgenic response.
- Common treatment includes sugar starvation, temperature stress, gamma irradiation, centrifugation or the use of chemicals like colchicine.
- Type of stress and time of application may vary among different species.
- Temperature shock is most commonly used. Whereas cold stress is useful in Indica rice, heat stress is useful in capsicum.
Origin of Androgenic Plants
Induction
- For most species a suitable stage for the induction of androgenesis lies between just before to just after pollen mitosis.
- During this phase of development the cells are non-committal in their developmental potential because most of the sporophyte-specific gene products are eliminated from the cytoplasm before meiosis and the gametophyte- specific genes are generally transcribed after pollen mitosis.
- After the first mitotic division the cytoplasm gets populated with gametophytic information and it gradually becomes irreversibly programmed to form male gametophyte.
- Stress induction at this stage can mask the gametophytic programme and induce the grains to switchover from gametophytic mode to sporophytic mode of development.
- Treatments such as temperature shocks (high or low), high osmolarity, and starving the grains of sugar or other nutrients are required to induce or promote the induction of androgenesis.
- The first cytological changes associated with the switch to sporophytic development is the loss of vacuole, the movement of the asymmetrically placed nucleus to the central position due to apparent loss of microtubule cytoskeleton investing the nucleus, and the appearance of starch containing plastids and globular domain within the cytoplasm.
Early Segmentation of Microspores
Based on the few initial divisions in the microspores four modes of in-vitro androgenesis have been identified:
1.Pathway I:- Both daughter cells (no distinction between veg and gen cell) contribute to sporophyte development , (Brassica sp.).
2.Pathway II:- Only Vegetative cell participate in development (capsicum, barley, tobacco etc.).
3.Pathway III:- Only generative cell participate in development (Hyoscayamus niger).
4.Pathway IV:- Both Vegetative and generative cells participate in development (Datura sp.)
Regeneration of Plants
- Multicellular tissue is produced before the pollen wall burst open.
- Tissue mass is of irregular shape at this time.
- Gain a shape of globular embryo, & differentiation continue (Direct).
- In cereals, tissue become polarized before pollen burst, which opposite to the germ pore (Wheat or barley).
- Tissue may further proliferate to form callus before further differentiation (Indirect embryogenesis).
- Culture media components influence embryogenesis significantly.
Diploidization
- Haploids are sexually sterile.
- Spontaneous doubling of chromosome occur by fusion of nuclei after first androgenic division of the microspore (cereals like wheat, maize etc.).
- High temperature stress can also cause chromosome doubling at low frequency.
- Colchicine or trifluralin are used to induce chromosome doubling.
- Plant at 3-4 leaf stage, soaked in 0.5% colchicine solution for 24-48 h.
- Washed thoroughly and allowed to mature.
- Concentration of colchicine and application time varies among different plants.
Applications of Androgenesis
- Haploids or double haploids are complete homozygous system and play important role in genetic studies.
- Haploids is an ideal system to detect and study recessive mutations.
- Gametic variations because of meiotic recombinations can be studied.
- An excellent model to study induction of embryogenesis and embryo development from haploid microspores.
- Can help to increase the frequency of desirable male plants
- Asparagus officinalis, male inflorescence having low fiber are preferred.
- Anther cultured homozygous males (YY) when crossed with females (XX), all the F1 progeny are males.
- Fertile homozygous double haploid lines can be produced in self-incompatible plants.
- Double haploid populations are ideal for genetic mapping. Can be used to develop linkage maps of molecular markers.
- These are very helpful in genomics studies and identification of major genes.
- Double haploid can be released as cultivars.
- Useful for true breeding diploid transgenics.
- It help in breeding hybrids or cultivars by reducing the time required for cultivar development.
References
- Surajit Das, Bhakti Patel, Chapter 28 – Marine resources and animals in modern biotechnology, Editor(s): Ashish S. Verma, Anchal Singh, Animal Biotechnology (Second Edition), Academic Press, 2020, Pages 567-591, ISBN 9780128117101, https://doi.org/10.1016/B978-0-12-811710-1.00027-6.
- Schwander T, Oldroyd BP. Androgenesis: where males hijack eggs to clone themselves. Philos Trans R Soc Lond B Biol Sci. 2016;371(1706):20150534. doi:10.1098/rstb.2015.0534
- Introduction to Plant Biotechnology, Third Edition, H.S. Chawla, ISBN 978-1-57808-636-8
- Androgenesis – Wikipedia
- Wang, Qing & Ran, Yidong & Yu, Bin & Chen, Xiaoyan & Wang, Di. (2014). Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch). In Vitro Cellular & Developmental Biology – Plant. 50. 525-533. 10.1007/s11627-014-9610-8.