The following points highlight the top five applications of pollen biotechnology: 1. Overcoming Pollination Constraints for Optimization of Crop Yield 2. Developing Effective Pollination Control System for Commercial Production of Hybrid Seeds 3. Overcoming Cross ability Barriers to Transfer of Useful Genes to Crop Species and Others.
Application # 1. Overcoming Pollination Constraints for Optimization of Crop Yield:
In majority of crop plants, seeds and/or fruits are the economic products. Pollination is a prerequisite for effective fruit- and seed-set. Most of the crop species, except cereals, are cross- pollinated.
Pollination of crop plants is often a major constraint due to one or more of the following factors:
(i) Reduction in native pollinator populations due to habitat degradation of pollinators.
(ii) Extensive use of agrochemicals like pesticides, insecticides and herbicides with drastic effects on native pollinator population.
(iii) High density of plants in monoculture cropping system limited the availability of native pollinators.
(iv) Crops introduced in regions where natural pollinators are absent.
Insufficient pollination causes low yield in many crops and orchard species. The proper management of pollinators can overcome pollination constraints, and will improve the yield.
1. Enhancing populations of native pollinators through habitat management:
The most important approach is to enhance local population of native pollinator species through habitat management. Habitat management is found to be very useful to increase native pollinator populations where nest sites availability is a limiting factor.
The practice of maintaining uncultivated strips along field margins or of providing artificial nesting sites increases population of native pollinators. The use of permanent nest boxes increases population of pollinators. The local population of bumble bees could be increased either by capturing queens in artificial nests, or moving them to a target crop.
2. Use of commercially managed pollinators:
The most effective way to overcome the pollination constraints is to use of commercially managed pollinators. For the purpose of commercial crop pollination, a number of social species of bees including honey bees and bumble bees, etc. is used. Honey bees are most useful pollinator, because of their large foraging population, transportability, year round availability and varied dietary preference in a wide range of crops.
Bumblebees are also found as pollinators in crop pollination for their large size, long-tongue length, ability to vibrate flowers requiring “buzz-pollination” and ability to fly at relatively low temperature or ability to fly in enclosed spaces.
But the use of bumble bees is restricted to high-value greenhouse crops, because of the technical difficulties and high costs associated with their management. Several solitary species of bees are also used for successful crop pollination, including alkali bee, leaf cutter bee, orchard bee, etc. These species live in a close association and reproduce in artificial nesting sites.
Pollination of the target crop may be decreased if neighbouring non-target crops and native plants provide greater nectars or pollen for the pollinators.
These negative impacts of floral competition on pollinator on a target crop can be mitigated by the following methods:
a) Altering the visiting time of bees to the crops.
b) Rotating or replacing colonies on the crop.
c) Temporal or spatial isolation of crops.
d) Improving crop attractiveness through crop breeding.
e) Using attractant sprays (dilute solution of pheromones, synthetic plant volatiles isolated from nectar or pollen, sugar syrup etc.) on target crop.
f) Training bees for making them attractant to crop odors.
g) Lowering the attractiveness of non-target plants.
Application # 2. Developing Effective Pollination Control System for Commercial Production of Hybrid Seeds:
Development of hybrid seed technology for commercial crop production is found to be one of the most important advances in the history of agriculture.
The pre-requisite of application of hybrid seed technology is the presence of hybrid vigour which refers to an increase in vigour and productivity of the hybrid compared to its parent. This method has been used in all corn growing countries with great success in production of hybrid seeds.
The commercial production of hybrid seeds in any crop species requires the following:
a) Development and maintenance of inbred lines.
b) Prevention of self and intra-line pollination in hybrid seed production.
c) Effective cross-pollination between two inbreed lines.
The success of production of hybrid seeds through hybrid seed technology implicates high cost and dependence on the technology. The tremendous success achieved in the production of hybrid maize, has lead to the use of such technology in many other crops, like sorghum, pearl millet, sugar beet and in many vegetable crops like, tomato, onion, brassicae, etc.
Now-a-days many pollination control systems are available for commercial hybrid seed production.
They are:
a) Use of Cytoplasmic male sterility (CMS).
b) Use of Genie (Nuclear) male sterility (GMS).
c) Use of Self-incompatibility (SI).
d) Use of pollen sterility induced through chemical hybridizing agents (CHAs).
e) Use of pollen sterility induced through recombinant DNA (rDNA) technology.
1. Use of cytoplasmic male sterility (CMS):
Plants that do not produce viable or functional pollen are male sterile. If such male sterility is exclusively maternally inherited, it is called Cytoplasmic male sterility. CMS is a convenient method for production of commercial hybrid seeds in plants.
This system consists of a male-sterile female “A-line”, a male-fertile maintainer “B-line” and a male- fertility restorer “R-line” (Fig. 9.1). Here the progeny shows no segregation into fertile and sterile plants, thus no rogueing of fertile plants is required in seed production plots.
CMS has several origins. They may be originated through intergeneric crosses, interspecific crosses, intraspecific crosses and mutagen or antibiotic effects on cytoplasmic genes. The genetic determinants of CMS are located in the mitochondria and nuclear genes controlling the expression of CMS. The restorer genes temporarily suppress male sterility.
Once the appropriate A-lines, B-lines and R-lines are produced, commercial seed production is done following two stages namely:
(i) The maintenance stage, and
(ii) The crossing stage.
In maintenance stage the seed quantities of the A-line, the B-line and the R-line are increased, while in crossing stage the production of hybrid seed using A line and R-line parents is done. As the maintenance stage is controlled by the originator of the hybrid, thus relatively small quantities of seeds of the A-line, B-line and R-line are required for one hybrid.
The production of hybrid seeds using CMS has been extensively done in many crop plants, e.g., maize, sorghum, pearl millet, sunflower, carrot, onion, Petunia, etc. However, many CMS systems have limitations, thus it is difficult to use them as pollination control systems in the production of commercial hybrid seeds.
The limitations include:
(i) Enhancing disease susceptibility.
(ii) Instability of male sterile and/or male fertile restored line due to environmental conditions.
(iii) Pleiotropic negative effects in the CMS cytoplasm in relation to agronomic quality performance of plants.
(iv) Insufficient nectar production affects cross-pollination in a few crops.
2. Use of genie male sterility (GMS):
Nuclear encoded, genie male sterility is reported in nearly every major crop species. GMS can be induced through mutations in any one of a number of genes controlling pollen and/or stamen development. The labour involved intensive process of flower emasculation is not needed in GMS, thus male- sterile plants of monoecious or hermaphrodite crops can be used in hybrid process. GMS is expressed under a homozygous recessive condition, hence the restorer line has to be heterozygous.
When A-line is crossed with 13- line, the progeny produced are 50% pollen fertile and 50% pollen sterile, here seeds are collected from male sterile plants to maintain the A-line. Thus, maintenance of GMS line involves mechanical removal of male fertile plants in the progeny (Fig.9.2).
Several proposals have been put forward to alleviate the problem of the removal of male fertile plants produced from the cross between homozygous A-line and heterozygous B-line.
Some of the proposals are:
(i) Identification of marker genes:
The suggestion is to identify marker genes that are closely linked to ms genes and are expressed in vegetative parts such as seed colour and shape, leaf or stem pigmentation, nature of trichomes. Such marker genes would help to identify the ms plants at an early stage.
(ii) Environmental/Hormonal induction of male fertility:
The ability to manipulate male fertility in GMS lines by environmental or hormonal treatment (Fig.9.3) is another desirable approach in hybrid seed production.
(iii) Vegetative/micropropagation of female lines:
Clonal micropropagation of GMS lines through vegetative propagation has been suggested for some ornamental and vegetable crops where vegetative propagation is very common.
3. Use of self-incompatibility (SI):
Self-incompatibility (SI) is defined as the inability of a fertile hermaphrodite seed plant to produce zy gotes after self-pollination. It leads to obligate outbreeding and also helps in maintenance of heterozygosity within species. There are two types of SI, viz., gametophytic self-incompatibility (GSI) and sporophytic self-incompatibility (SSI).
GSI is a common outbreeding mechanism occurs in more than 60 angiosperm families, Solanaceae in particular. GSI is governed by a single highly polymorphic locus. Pollen carrying an S-allele identical to one of the two alleles located in pistil is prevented from effective fertilization.
In SSI, pollen phenotype is determined by the genotype of the mother plant and the phenotype of the pollen is determined by dominance interactions. The number of S-alleles at the S-locus is unusually large, for example 34 in Raphanus, 60 in Brassica oleracea.
Use of SI is found to be more advantageous over CMS and GMS in production of hybrid seeds.
Advantages:
i) No need to develop maintainer or restorer lines for SI.
ii) Use of SI reduces the cost of hybrid seed production as hybrid seeds can be harvested from both the parents.
iii) No pollination constraints are found as both lines are pollen fertile with their natural ability for cross-pollination.
SI posses some problems related to developing and maintaining pure lines. So it is necessary to manipulate SI systems so that they are sufficiently stringent to use in the breeding of F1 hybrids.
Several methods are now available to overcome SI:
i. Induction of mutations.
ii. Induction of autotetraploidy.
iii. Bud pollination.
iv. Delayed pollination.
v. Hot water/high temperature treatment.
vi. Application of growth substances.
vii. Use of mentor pollen.
viii. Placental pollination.
ix. High CO2 concentration (3-6%).
x. Treatment of flowers with high CO2 and humidity.
xi. Treatment of stigma with NaCl (1.5% – 3%) before or after pollination.
xii. Treatment of stigma with lectins or pollen with sugars before pollination.
Though most of the crop species are self- compatible, many wild relatives are found to be self-incompatible. So it is not easy to transfer of SI from SI species to self-compatible cultivars. Recently S-genes have been partially characterized and this opens up the possibility of transferring the S-gene through recombinant DNA technology.
4. Use of pollen sterility induced through chemical hybridizing agents:
Chemicals capable of selectively inhibiting pollen development by blocking male fertility are known as chemical hybridizing agents (CHAS) which are used for the large-scale commercial production of hybrid seeds. Most CHAS are applied at certain critical stages of male gametophytes development.
The mechanisms of action include:
(i) Male gametocides or inhibitors of anther development,
(ii) Selective transport of toxic or growth inhibitory substances to the anthers, and
(iii) Metabolic detoxification of toxic or growth inhibitory substances after the suppression of male fertility.
Several CHAs have been reported for the commercial production of hybrid seeds.
They are classified as:
(i) Inhibitors of pollen fertility
1. Proline analogues
b. Methanoproline (cis-3,4- methylene-S-proline)
c. Azetidine-3-carboxylic acid.
(ii) Inhibitors of microspore development
1. Phenyl Pyridazones
a. Fenridazon (RH-0007), a phenyl- substituted pyridazone carboxylate.
2. Phenylcinnoline carboxylates
a. SC 1058[1-(4 trifluoromethyl- phenyl)-4-oxo-5-fluorocinnoline – 3-carboxylic acid]
b. SC 1271 [1- (4/-chlorophenyl)-4- oxo-5-propoxy-cinnoline-3- carboxylic acid]
c. SC 2053 [1-(4/-chlorophenyl)-4- oxo-5-methoxyethoxy-cinnoline-3- carboxylic acid]
d. MON 21200 [Pyridine monocarboxylic and benzoic acid analogue]
e. Copper chelators
f. Ethylene
(iii) Disrupt floral development
1. LY195259 [5-(aminocarbonyl)-1-(3- methyl-phenyl)-1 H-pyrazole-4- carboxylic acid]
5. Use of pollen sterility induced through recombinant DNA (r- DNA) technology:
In recent times many pollen-specific and anther-specific genes have been discovered. Some anther-specific genes are expressed in diploid tapetum, but many others are confined to haploid pollen grains. These discoveries have opened up a new field to use recombinant DNA (r-DNA) technology for induction of pollen sterility through breakdown of developing pollen by tagging a cytotoxic gene under the control of a tapetum or pollen-specific promoter.
The basic concept is to target the expression of a gene encoding a cytotoxin by placing it under the control of an anther (tapetum) – specific promoter. This is done by constructing a chimeric gene containing 5′ regulatory (promoter) region of the TA29 gene, characterized by its tapetal-cell specificity and a gene encoding ribonuclease.
One of the ribonuclease genes, RNase T1 was chemically synthesized from Aspergillus oryzae, and the other was natural gene, called barnase from the bacterium Bacillus amyloliquefaciens which produce RNase.
Both chimeric TA29-RNase genes were introduced into many transformants. RNase production led to degeneration of tapetal cells, arrest microspore development and male sterility in many crops like Brassica napus, maize and several vegetable species.
It was observed that absence of chimeric genes led to pollen fertility. Some investigators have used antisense RNA technology or cosuppression of endogenous genes which are found to be essential for pollen development.
In connection with the commercial production of hybrid seeds by the use of pollen sterility developed through r-DNA technology, suitable restorers are needed. The first genetically engineered RF (restorer) gene was reported by Mariani (1992). Barstar gene produces barstar protein which is a specific inhibitor of barnase (one of the RNase used to engineer male sterility) under the control of the TA29 gene promoter.
Expression of barstar gene under the same promoter as the barnase gene was done to ensure that it would be activated in tapetal cells and would accumulate in amounts at least equal to barnase. The majority of crosses between TA29-Barstar and TA29-Barnase plants produced progeny with both chimeric genes that are male fertile (Fig.9.4).
The possibility for producing 100% male sterile populations was demonstrated by Mariani (1990) who linked the dominant TA29-barnase male sterility gene to a dominant marker gene, the 35S bar that shows tolerance to the herbicide glufosinate ammonium.
The application of herbicides to the plants of the female parent at seedling stage will eliminate 50% male fertile and herbicide sensitive plants (Fig.9.5).
Application # 3. Overcoming Cross ability Barriers to Transfer of Useful Genes to Crop Species:
In conventional breeding programme the useful genes are transferred to crop species from other accessions or species through normal hybridization process. In recent years, crop improvement programmes highlighted exclusively on increasing yield and improving quality and genetic uniformity.
In addition to the improvement in yield and quality, the farmers are also interested to develop varieties having low requirement for chemical fertilizers, herbicides, pesticides and are resistant to biotic and abiotic stresses such as diseases, pests, salinity, drought, etc. The desired genes of the cultivated crop species are no longer available due to constant loss of genetic diversity.
However, a large number of wild relatives of crop species are the good source of such desirable genes. The use of pollen for screening desirable genes has come up in recent times and has many advantages. Pollen grain is a single haploid cell that representing a male gametophytic generation.
A single plant produces huge amount of pollen grains. It is very easy to collect huge amount of pollen grains in uncontaminated condition. The use of pollen as vector for screening desirable genes is found to be more effective which reduces the time and cost of screening.
It has been reported that a large number of genes are pollen-expressed and about 60% of them are also expressed in sporophytic tissue. The positive correlation between performance of pollen and that of the parent sporophyte under a range of stresses indicates the adaptability of genes that are expressed in pollen as well as the sporophyte. Several pollen biotechnological approaches have been used to find out the major step or constraint associated with conventional breeding program (Table 9.1).
1. Use of pollen for genetic transformation:
A high plant regeneration frequency is needed for transfer of successful genes and the recovery of transgenics. Pollen embryos generally show a high regeneration potential.
A number of gene transfer techniques have been successfully utilized in the transformation of pollen embryo. A number of techniques have been adopted for gene delivery into pollen or microspores. These include imbibition of pollen with DNA, Agrobacterium-mediated transformation, electroporation and polyethylene glycol-mediated transformation.
(i) Agrobacterium-mediated gene transfer technique has been used for the recovery of fertile homozygous transgenic plants. But the success was not achieved in transformation of tobacco pollen by co-cultivation with Agrobacterium tumifaciens. Because, the hydrated pollen grains have high nuclease activity, which inhibit the introduction of exogenous DNA into the pollen.
(ii) The use of electroporation and Polyethylene glycol (PEG) are done to facilitate direct DNA uptake by microspore to facilitate transient gene expression, but stable transformants are not recovered. Moreover, it is difficult to isolate protoplast from pollen grains, except in some liliaceous members, thus electroporation cannot be a suitable technique.
(iii) In recent times, particle bombardment method has been of great use for transfer of gene to pollen grains. There are two strategies applied to the use of particle bombardment to achieve pollen transformation for crop improvement.
Firstly, transgenic seeds are formed upon pollination with transformed pollen grains. Secondly, the transgenic haploid plants are produced directly from transformed pollen grains through in vitro culture of pollen (Fig.9.6).
There are several successful gene transfer into pollen by particle bombardment. LAT 52 promoter from tomato trigger the expression of GUS (β-glucuronidase) reporter gene in tobacco and tomato pollen. The same promoter has been used successfully to trigger the expression of foreign gene in the pollen of Nicotiana glutinosa and Nicotiana rustica and peony.
Two anther-specific promoters from tomato (LAT56 and LAT59) and one pollen- specific promoter (PA2) from Petunia have been used to trigger the expression of gene in pollen of tobacco. Zm 13 promoter from maize has been reported to express in the pollen of maize and Tradescantia. Several anther- and pollen-expressed promoters have been identified and used to trigger the expression of genes in pollen by particle bombardment.
2. Techniques for overcoming pre- fertilization barriers:
Interspecific hybridization is one of the important techniques for crop improvement. A range of techniques are now available to overcome the pre-fertilization barriers at different levels. Thorough studies on the details of the barriers would assist the breeder’s ability to apply the most useful techniques to overcome hybridization barriers.
The various methods are mentioned below:
(i) Genetic variation in interspecific cross-ability:
The interspecific cross-ability between two different species is obtained by genetic as well as environmental factors. So it is important to test different feasibilities of the parents in hybridization programme.
Unilateral incongruity is the phenomenon where a cross is found to be successful only in one direction, so that the reciprocal cross is unsuccessful. It has been observed that crossing barriers can be overcome using unidirectional cut-style pollination.
(ii) Use of mixed and mentor pollen:
Mixed pollen is made from a mixture of compatible and incompatible pollen. Mentor pollen is compatible pollen which is genetically inactivated by irradiation but still capable of germination. The use of mixed pollen and of mentor pollen, mixed with incompatible pollen has been reported to overcome inhibition on the stigma and/or in style in many plant species.
(iii) Influence of environmental condition:
The optimal level of receptivity of the stigma varies from several hours to more than one week. A positive effect of high temperature to overcome self-compatibility and incongruity has been established in breeding programmes either by heating the style or by pollinating at high temperature.
(iv) Style and ovary manipulation:
Pollen tube growth inhibition in the style can be overcome by manipulating style and ovary. This technique has been observed in Fritillaria. The manipulation involves removal of the stigma and a part or whole of the style and the pollination is done in cut end. This technique is called ‘stump pollination’ or ‘cut style’ or ‘amputated style’ pollination.
(v) Chemical treatment:
Administration of growth hormones (auxins, cytokinins, Gibberellins) to the pedicel or ovary during or soon after pollination improves fruit and seed. In some interspecific crosses, treatment of the stigma with organic solvents (hexane, ethyl acetate) before pollinaton is found to be effective to overcome pre-fertilization barriers. In many cereals, immuno-suppressors like salicylic acid, acriflavin and amino-n- caproic acid have been used toproduce hybrids.
3. Techniques for overcoming post- fertilization barriers:
There are several post-fertilization barriers for hybridization and these include, (i) non- viabilities of hybrid embryos, (ii) failure of hybrid to flower, (iii) hybrid sterility, (iv) lack of recombination, (v) hybrid breakdown in F2 or later generations.
The cultures of ovary, ovary-slice, ovule and embryo are found to be the important aspects to overcome post-fertilization barriers. Ovary culture has been successfully applied in breaking post-fertilization barriers in many crop species like Brassica, Lilium, Tulipa, Phaseolus and Eruca-Brassica hybrids. Ovary-slice culture has been applied for the production of inter-specific Lilium hybrids.
Ovule culture is an easy and fast technique which can be applied in crops where fruits are generally aborted. This technique has been successfully applied in Lycopersicon, Nicotiana, Vitis and Alstroemeria, etc. for overcoming post- fertilization barriers.
Embryo culture has been successfully applied in crosses where pollinated flower stay on the plant for a prolonged period before abscission. This method has been successfully employed in Allium, Freesia, Lilium, Lycopersicon, Solanum etc.
4. Pollen storage to overcome physical barriers:
Pollen grains have long viability and can be stored for a long time through various modern techniques.
Pollen storage has a wide range of application in the different areas of reproductive biology to fulfill the following needs:
(i) Hybridizing plants that show non- synchronous flowering.
(ii) Promoting supplementary pollination for improving yields.
(iii) Eliminating male lines continuously in breeding programme.
(iv) Providing a continuous supply of short-lived pollen.
(v) Assisting the exchange of international germplasm.
(vi) Confirming year round availability of pollen without using nurseries or other means for plant growth.
(vii) Ensuring the study of pollen biology.
It is very essential to establish “Pollen banks” where pollen grains of a desired species would be preserved as genetic resource for breeding programme. Several methods have been put forward to assess various storage conditions for extending pollen storage. Now it is possible to store pollen grains of a large number of species, thus several ‘pollen banks’ are formed. Pollen grains can be stored both in short term and long term basis (Table 9.2).
The important methods of pollen storage are mentioned below:
(a) Short-term storage of pollen
(i) Storage under low temperature and low humidity:
Pollen grains can conveniently be stored in a glass or plastic vials. The unsealed containers are kept into desiccators containing suitable dehydrating agents (dried silica gel, saturated solutions of different salts or H2SO4) to maintain in low RH (<10%). Then the sealed desiccators are kept in refrigerator or deep freezer. In this method pollen can be stored for weeks or months. But the storage under sub-freezing condition (ca -20°C) will be very effective for storage of pollen for more than a year.
(ii) Storage in organic solvent:
This is a very simple method by which pollens are dried over silica and stored in organic solvents and maintained in refrigerator or deep freezer. Pollen stored more effectively in non-polar organic solvents like hexane, cyclohexane or diethyl ethar which retained their viability and showed very little leaching of phospholipids, sugars and amino acids in the solvent.
(b) Long-term storage of pollen:
(i) Storage of freeze or vacuum dried pollen (Lyophilization):
The freeze- and vacuum- dried pollens are stored at subzero temperature (-60°C to -80°C). In freeze-drying the pollen grains are freezed at subzero temperature with gradual removal of water under sublimation, while for vacuum drying pollens are exposed to simultaneous cooling and vacuum drying.
Pollen grains stored in these methods exhibit no difference in response to storage. The freeze/vacuum dried methods are found to be effective for storage of pollen of various species, except for cereals, for more than a year (Table 9.2).
(ii) Cryopreservation:
This is a much simplified technique by which pollen grains are dried by using a ‘Pollen dryer’ containing air of 20°C and 20-40% humidity to bring their water content below a threshold level and stored in liquid nitrogen (- 196°C). This method is very effective for storage of pollen of a number of species, including cereals, even for over 10 years.
Successful storage of pollen grains of a large number of species has been done by various workers. A list of such plants has been given in Table 9.2.
5. Multiplication of hybrids:
A large number of hybrids are needed for induction of amphidiploidy to restore fertility and also to raise backcross progeny. Hybrids are needed for morphological, cytological and biochemical studies.
In-vitro culture technique is very useful for multiplication of hybrids through culture of shoot tip or single node segment. Callus culture is another conventional method for multiplication of hybrids. Callus may be induced from hybrid embryo or hypocotyl segment and subsequent regeneration of plantlets are done through shoot or root generation or through somatic embryogenesis.
6. Identification of hybrids at seedling stage:
Interspecific crosses may not always produce a hybrid due to incompatible pollination. So the hybrid nature of the resultant plant from the interspecific cross has to be confirmed.
The identification of a hybrid plant through morphological and cytological studies is possible only at its adult stage. Though it is a conventional method but it is time consuming. If the resultant plant turns out to be non-hybrid, there would be wastage of money as well as time. So it is important to find out a hybrid at an early (seedling) stage.
The usual and effective way to identify hybrid is to use phenotypic markers that are expressed at seedling stage. In other cases, a hybrid can be identified by studying the isozyme patterns or by molecular techniques such as oligonucleotide finger printing, RFLP, RAPD and species- specific repetitive DNA sequences.
Application # 4. Induction of Haploids from Pollen Grains and their Utilization:
Haploids are the sporophytic plants which have a gametophyte chromosome number (n). After the discovery of first natural haploid in Datura in 1921, several natural haploid plants have so far been reported in a large number of species.
Scientists, especially plant breeders are interested to induce natural hybrids through the various techniques for their use in crop improvement. The first successful induction of haploids (Pollen embryos) in cultured anthers of Datura innoxia was made by Guha and Maheswari (1964, 1966).
Since then, several haploids have been made in cultured anthers/ microspores in a large number of species including crop plants. Now, this technique has widely been used for induction of haploids which is found to be an effective measure of plant breeding programme.
1. Production of pollen embryos:
Production of haploids from cultured anthers and microspores is one of the important areas of pollen biotechnology. Embryos that are induced from cultured anthers as well as microspores are developed from microspores and not from pollen grains.
In common term they are called pollen embryos. Initial success achieved in induction of pollen embryo was from culture of anthers, where anthers in suitable stages are removed from buds and cultured in a semisolid medium.
Anthers were also cultured in liquid medium on filter paper bridges. Even in grasses, inflorescence segment containing one or two spikelets were also cultured to get pollen embryos. Formation of pollen embryos is associated with two phases namely, induction phase and development phase.
It was found that anther tissue favours the induction phase but is often inhibitory for embryo development. This is perhaps due to the inhibitory substances from the degenerating tissues of the anther.
Sometimes anther tissue forms callus and competes with embryo/callus derived from pollen. It has also been noticed that microspores in cultured anthers give rise to a heterogenous callus instead of embryos. But culture of microspores, instead of anthers eliminates those limitations. Remarkable success has been achieved in culture of microspore for induction of pollen embryos.
In initial experiments, microspore culture showed the combination of both anther and microspore culture. Later, Sunderland and Roberts (1977) proposed a ‘Shed pollen culture’ technique in tobacco in which pretreated anthers are isolated and then floated on shallow liquid medium with high osmoticum to allow anther wall dehisce and release of pollen to produce mature embryos even after removal of original anthers. It has been shown in some species that induction of embryos is enhanced by centrifugation of anthers before microspore isolation for production of pollen embryos.
2. Advantages of microspore culture compared to anther culture:
(i) It is easy to isolate microspores, which are less time consuming compared to isolation of individual anther.
(ii) The culture density for optimum response can be maintained with isolated microspores.
(iii) Microspore populations can be enhanced by different techniques like cell sorting or gradient centrifugation. These techniques are not applicable for anthers.
(iv) The every stage of embryogenesis can easily be measured with the isolated microspore system, and the factors associated with every stage can be identified.
(v) The isolated microspore system is well adapted with biochemical and physiological studies.
(vi) If the frequency of embryogenesis is low, callusing of the anther walls overgrow microspore-derived embryos and subsequently suppress their development. In isolated microspore culture, this problem can be avoided.
(vii) The isolated microspore culture system is better adapted for gene transfer techniques and mutagenesis in comparison to anther culture.
3. Optimum conditions for induction of pollen embryos:
(a) Genotype:
The genotype of the donor plant influences the responsiveness of cultured anthers/microspores. It has been observed that wheat pollen embryogenesis is controlled by at least three dominant genes.
(b) Physiology of donor plant:
It has been established that the embryogenic response of the cultured anthers/microspores is greatly influenced by the age and physiological status of the donor plant. In general, plants growing at lower temperature respond better that those growing at higher temperatures.
(c) Composition of medium:
The induction of pollen embryo is dependent on the composition of media. It has been observed that exogenous hormones, specially auxins, are required for induction of haploids in most cereals, Medium containing coconut water, potato extract, yeast extract with higher concentration of hormone induced callus. Even a hormone-free medium also induced pollen embryo.
Carbohydrates play an important role to induce pollen embryo. It has been observed that the frequency of embryo induction increases with increasing concentration of carbohydrate. Both sucrose and maltose are found to be effective in different species. The other factors like pH, nitrogen source, anti-toxic metabolites and jelling agent of medium are also responsible for induction of pollen embryo in different species.
(d) Stage of microspores at culture:
The divisional stage of microspore at culture is an important condition for induction of embryogenesis in most of the species. It has been observed that microspores cultured at late uninucleate or early binucleate stage are more effective for induction of embryogenesis at earlier or later stages.
(e) Pretreatment of anthers/flower buds:
Temperature shock for different periods alters division of microspores from asymmetric to symmetric, thus enhances induction of pollen embryos in several species.
Pretreatment of anthers/flower buds at low temperature (3-5°C) for 48 hours is effective to induce pollen embryos in many species like Nicotiana tabacum, Datura metel, D. innoxia, Brassica napus, etc. Similarly, pretreatment with high temperature (30-45°C) is also found to be effective in increasing embryogenesis, especially in Brassica spp., rice, etc..
4. Developmental pathways of embryogenesis:
1. Pollen dimorphism:
There is a dispute regarding the nature of microspores that form pollen embryo. In the first view, the scientists claimed that the embryogenic pollen grains are morphologically distinct from gametophytic pollen in anthers (dimorphic pollen). Embryogenic pollen grains are smaller with thin exine showing poor affinity for cytoplasmic stains.
While, the gametophytic pollen grains are larger with thick exine showing strong affinity for cytoplasmic stains. After induction, embryogenic pollen grains develop pollen embryos, while normal (gametophytic) pollen grains degenerate.
In some species, pollen grains do not show any dimorphism, but under inductive condition pollen shows dimorphism. In the second view, pollen grains in anthers are morphologically alike. Under inductive conditions, all microspores become potentially embryogenic and form pollen embryo.
2. Developmental pathways:
The development of androgenic plantlets show two different patterns:
(i) Pollen embryos develop directly from microspores and give rise to plantlets, and
(ii) Microspores develop into callus, and subsequently plantlets are produced from the callus through organogenesis or embryogenesis.
Embryos developed from microspores callus show polarity from the beginning where distal pole is free, while proximal pole is attached to the non-embryonic cells.
Sometimes a prominent suspensor like region is seen. The polarity is not apparent during initial stages of division in embryos deriving directly from the pollen without callusing. It has been evidenced that the plasma membrane proton pump (H+-ATPase) associated with the membrane of Arabidopsis microspores plays an important role in the formation of pollen grains from microspores.
The development of pollen embryos shows two different ontogenic pathways:
(i) First ontogenic pathway:
Firstly, microspore divides symmetrically to produce two equal cells and the embryo or callus is developed from the activity of both cells (Fig 9.7). This ontogenic pathway is very common in several species like Lycopersicon, Atropa etc.
(ii) Second ontogenic pathway:
Microspore divides asymmetrically to form a vegetative cell and a generative cell like the normal gametophytic development of pollen (Fig 9.7). Then, embryo/callus is derived from the activity of only vegetative cell or only generative cell or rarely from both. The development of embryo/callus from vegetative cell is very common in a number of species like Nicotiana, Lolium, Festuca, Datura, Triticum, etc.
In this pathway generative cell does not divide further or rarely follows a few divisions before degeneration. Pollen embryo originates from generative cell has been observed in Hyoscyamus niger, while the embryo derived from the activity of both vegetative and generative cells has been reported in Datura innoxia.
5. Utilization of haploids (Pollen embryos):
1. Breeding and genetic studies:
The most obvious advantage of pollen embryos to crop improvement is to create homozygous plants through chromosome doubling which reduces the need to conduct time consuming inbreeding phytic and sporophytic (embryogenic) pathways of cycles.
In this method, all functional genes are expressed from double haploids because of having all the homozygous loci. Commercial application of this technique has been done in wheat and tobacco. The value of double haploids is noticed in some outcrossing populations like Brassica oleracea and B. napa where this method is successfully applied to achieve homozygosity.
2. Mutation and selection:
It has been most obvious advantage that most of the embryogenic microspores are subjected to in vitro selection pressure and can be mutagenized in a relatively small space.
In pollen-derived embryo system, mutants can easily be isolated and selected, because all recessive characters are expressed which is a close parallel to microbial system. This technique has been successfully applied to isolate the herbicide tolerant Nicotiana tabacum and Brassica napus plants through in vitro mutagenesis of isolated microspores obtained from pollen embryo.
The gametoclonal variations among pollen embryo will lead to select the useful traits without the need for mutagenesis. This has been successfully applied to select the variants for disease resistance, increased alkaloid content and increased protein levels.
3. Gene transfer:
Pollen embryos are potentially useful as recipients for foreign genes. Several gene transfer techniques have been adopted in the transformation of pollen embryos, of which particle bombardment technique is conventionally used.
4. Biochemical and physiological studies:
Pollen embryos are ideal material for biochemical and physiological studies of embryogenesis in various areas like chilling tolerance, metabolism of chlorophyll during seed degreening and glucosinolate metabolism. Storage lipid and protein biosynthesis have extensively been studied in Brassica pollen embryos. These embryos contain high amount of lipid biosynthesis enzymes and are useful for in vitro screening for oil quality.
5. Artificial seeds and germplasm storage technology:
The production of artificial seeds using somatic embryos is a modern biotechnological method that involves (i) embryos maturation, (ii) development of tolerance to desiccation. This technology has also been applied to the pollen embryos Pollen embryos are more advantageous for artificial seed production, because (i) microspore derived embryo is much more uniform compared to the somatic embryo, (ii) it is possible to diplodize microspores before embryo formation to recover homozygous embryos. A large number of pollen embryos are the potential source of germplasm for crop improvement which are derived from genetic re-combinations and novel variations through the in-vitro culture or chromosome doubling.
Application # 5. Utilization of Pollen for other Purposes:
Pollen grains are exploited in basic research, because pollen is representing a generation and a haploid (gametophyte) plant can be made from single pollen. They are also used for commercial purposes as health food support as well as medicine. Pollen grains are also responsible to cause human allergy. So pollen is used for diagnoses and treatment of pollen allergy.
1. Pollen as health food:
Since pre-historic times, pollen has been used as food supplement. Pollen grains are rich in proteins, sugars, minerals and vitamins (especially B-complex with low values of fats, sodium and fat-soluble vitamins like D, K and E.
Pollen forms an excellent human food supplement because nutritional composition of pollen surpasses that of other foods commonly used by human being. So use of pollen as health food has gained importance and eventually several pollen-based industries has been developed.
Now-a-days pollen grains are sold in tablet or liquid form as a nutritional supplement. These foods are very popular among athletes and patients. Pollen grains are also used as food supplement to the diet of a variety of animals that shows beneficial effects.
2. Pollen as medicine:
Honey contains a considerable amount of pollen grains, though honey is made up largely of nectars from flower. So pollen forms a critical component of the honey. The beneficial activity of pollen grains present in the honey has been established. Some of the beneficial properties of bee pollen are: antibiotic qualities, intestinal functions, combat respiratory problems, balance endocrine system.
3. Pollen allergens for diagnosis and therapy:
It is now well established fact that pollen grains are responsible to cause human respiratory allergy. After maturity pollen get liberated and dispersed by wind or animal vectors and remain suspended for sometime in the air. The atmospheric pollen grains are trapped either by using gravity slide method or by volumetric air samplers.
Identification of the dispersed pollen is mainly done on the basis of comparison with reference slides made from the local flora. In this connection a pollen calendar is to be formulated to render identification of airborne pollen grains with reference to their seasonal and diurnal variations.
The allergenicity tests of the pollen grains are done both by in vivo method (skin-prick test or patch test or scratch test) and in vitro method (ELISA, immunoblotting, immuno electrophoresis, etc.). An Atlas covering an illustrated account of airborne allergenic pollen is generally made which is found to be helpful for allergologists and clinicians for identification of the causative allergenic factors.
The respiratory allergy or asthma caused by pollen grains can now the tacked. To effect prevention, the following measures can be adopted:
(i) Eradication of generally offending plants and selecting trees for planting properly suited to the climate, but at the same time avoiding those which produce allergic pollen grains.
(ii) Removal of patients from place of habit to other places where such offending plants do not grow.
(iii) Clinical application: Antigenicity of different airborne pollen besides other inhalant allergens are tested by a standard clinical method on susceptible individuals. Desensitization, with vaccine prepared with allergenically significant pollen grains, produce appreciable improvement in susceptible patients. In this respect, characterization of different pollen allergens has been made for therapeutic use.