Plant Biotech Blog

C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

Biotic pollen vectors such as honey bees and bumble bees and some others have an important role in sustainable agriculture, but that has been exaggerated, romanticized and emotionalized by expansive claims by the environmentalists.  One such statement was attributed to Albert Einstein who reportedly said that ‘If there are no bees on the globe, then man would only have four years of life left.  No more bees, no more pollination, no more plants, no more animals, no more man’.  Without biotic pollen vectors certainly there would be problems with a few agricultural or some horticultural crops, but that would not be the end of the world.

The pollination behavior of the cereal and millet crop plants shows that most of them are highly self-pollinated or wind pollinated.  Biotic vectors do not visit these species.  In some other crop species biotic vectors that visit the flowers only take what they want such as nectar and/or pollen, and not necessarily pollinate. However, biotic vectors are important pollinators of a considerable number of species of fruit and vegetable crops and several wild species.

Relative antiquity of flowering plants and bees

The relative antiquity of biotic vectors and crops shows that they are not overly dependent upon each other.
The bulk of archaeological evidence indicates that cereals and pulses were in cultivation for about 6,000 to 7,000 years and the cucurbits, the oldest vegetable group, have been around for 5,000 years.

Molecular evidence based on chloroplast DNA sequences, supported by analyses of the nuclear genes encoding ribosomal RNA subunits, fixes the upper bound for the onset of flowering plants at 340 million years before present (mybp).  The dicot-monocot divergence, the major event in flowering plant evolution, has occurred at around 200 mybp.  Lineages of well defined dicots (the group of vegetables, pulses, tuber crops, etc.)  are dated around 170 mybp and the divergence of grass groups (rice, wheat, barley, maize, sugarcane, etc.) was dated around 100 mybp.

The fossil of Trigona prisca, a stingless honey bee, reported from New Jersey amber was dated around about 85 mybp.  This is a fairly advanced species, closely similar to modern neotropical species.  This is the oldest fossil bee known but with no evidence of any morphological evolution for the past 75 mybp.  Since the fossil is a worker, social organization had arisen by its time.

A phylogenetic analysis involving nucleotide sequences of five genes plus 101 morphological characters suggested an African origin for bees, which is not true for most of the crop plant species.

Comparative evidence indicates that honey bees appeared much later than the groups that gave crop plant species, though long before agriculture has originated about 10,000 years ago.  While bees and ancestors of crop plants shared the same environment for millions of years, along with thousands of other animal and plant species, their association often considered as co-evolution, is not so in the same intimate sense as that of pests and pathogens and their hosts.  Besides, crop plant species arose very rapidly during past 5,000 years through conscious human selection, the pace of which is no match to the much slower evolution of the bees and other pollinators by natural selection.  Considerable evidence indicates that there was no appreciable evolutionary progression in several groups of insects during the past 50 mybp or more.  Seasonality of flowering in crop plants, and largely shifting and nomadic cultivation till about the turn of the Christian Era (the plough came in around 100 CE), did not promote pollinator-crop dependence.  Throughout their evolutionary history bees continuously discovered suitable wild plants, including the ancestors of cultivated plants, for nectar and pollen, like the herbivores, plant pests and pathogens.  At the same time, both wild and cultivated plants had alternative means for pollination, just as they had before bees and other pollen vectors came onto the scene.

Pollination in Cereal Crops

The cereal and millet food crops of rice, wheat, corn, barley, oats, sorghum, pearl millet and finger millet all belong to the grass family.  They offer no incentives to biotic pollination vectors such as nectar, nutritionally rich pollen or have attractants like fragrances or bright colors.

The pollen of grasses are small (around 30 to 40 μm in diameter, more often smaller) smooth, dry and powdery, features ideally suited to be airborne.  They do not stick together, or to the body parts of the vectors.  They cannot be easily compacted either in the pollen baskets or the hives without additives.

It is difficult to distinguish pollen of one grass species from another under a light microscope as they have very few surface features to characterize them.   However, under a scanning electron microscope one may find features that help in distinguishing pollen of different species, and in some cases even pollen of different varieties.

The grass pollen have only a meager but physiologically functional pollen kit.  Grass pollen are very sensitive to temperature, sunlight and humidity.  At below 260 C and very high humidity anthers do not dehisce and at more than 320 C pollen viability suffers.

Grass pollen are trinucleate at the time of dispersal and so have notoriously short periods of germinability and viability.  The pollen of many grasses are difficult to germinate in the lab.

The grass crops are all largely self-pollinated.  Cross-pollination, to whatever extent that may occur, is by wind borne pollen, as biotic vectors do not normally visit grass inflorescences.

In the predominantly self-pollinated rice crop, the pollen are viable for less than 10 min, the stigma is receptive for about an hour and the florets close in less than 2 h.

Corn has unisexual inflorescences, the tassels (male) and cobs (female).  Corn pollen are among the largest (about 100 μm in diameter), rich in starch and are heavy.  The pollen settle more readily than windborne and the viability is less than 2 h.

When the pollen from the tassels of a corn plant reach the cobs below on the same plant, it amounts to self-pollination in genetic terms.

Pollination in Cotton

In cotton the anthers and stigmas are seated deeply in the bell shaped flowers.  Cotton pollen are about 50 μm in diameter, highly hydrated, heavy, spiny and sticky.  The floral structure and pollen features do not facilitate air lifting of pollen.

Over 80 per cent self-pollination occurs in cotton.  Several vectors visit the flowers but this does not ensure cross pollination.  On dissection immediately after they fed on cotton flowers none of 32 honey bees contained even a single pollen grain in their honey stomachs.  The bees drew the nectar but did not take pollen.

Pollination in Potato, Tomato and Aubergine

Potato (Solanum tuberosum), tomato (Solanum lycopersicum, Lycopersicum esculentum) and aubergine (egg plant, brinjal, Solanum melongena) are similar in floral structure and pollination biology.

The anthers of these species are hollow tubes that open by small apical pores, unlike in most other plant species where the anthers open dehiscing longitudinally to fully expose the pollen to the air and pollinators.

Solanum pollen are sticky and do not travel long distances, even if they become airborne.
When species of Solanum were introduced outside their native South American regions the original pollinators were not taken along to their new homes.  Hence, even varieties of these species are not normally cross-pollinated in nature.  Insects visit Solanum flowers but they can collect nectar without touching the anthers or stigmas, as the petals open flat out like the spokes of a wheel.

As potato is vegetatively propagated by the ‘eyes’ of the tuber (the farmer’s seed), pollination is not a matter of concern.  The breeders who are interested in the true fruits and seeds of potato hand pollinate the flowers.

In the wild state, tomatoes required cross-pollination.  Domesticated cultivars of tomato have been selected to maximize self-fertilization. Experimental studies with tomatoes have shown that pollinators such as bumble bees are ‘buzz pollinators’ which actually ‘sonicate’ the anthers causing the pollen to move out of the tubular anthers.  Gushes of wind, even artificial wind or cultured bumble bees can provide sufficient motion to produce commercially viable crops.

In cultivated aubergine the extent of self-pollination is over 90 per cent.

Pollination in Cultivated Brassicas

Most of the cultivated members of the mustard family (the Brassicaceae), such as Canola, oil rape, oil mustards, condiment and leafy vegetable mustards and turnip all belong to the genus Brassica.    Cabbage, cauliflower, knolkohl, broccoli, Brussels’ sprouts and related group of vegetables are different varieties of Brassica oleracea.  Radish (Raphanus sativus) also belongs to the same family.  Farmers had no difficulty in maintaining them distinct without loss of their identity, even when they are highly interfertile like the varieties of Brassica oleracea.

Wild species of Brassica and many other members of the family Brassicaceae have genetically determined self-incompatibility factors that prevent true self-fertilization.  However, during centuries of domestication and cultivation this has changed considerably to the extent that cross-fertilization is usually less than 30 per cent.

Brassica flowers are honey flowers, visited by bees, which may pollinate the flowers.  Pollen may also be airborne, much depending upon the temperature, rain or humidity, flowering stage and other related factors.

Pollination in Legume crops

The flowers of the legume crops such as pea, chickpea, soybean, and several others are intricate structures evolved to promote cross-pollination, but in practice they are self-pollinated, most often even before the flower opens and in the groundnut the flowers may not even open.

C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

Bees are heavily implicated in pollination of plants, more by popular belief than by rational science.  Honeybees actually need nectar and pollen from flowers for survival.

Nectar

Nectar, the sugary fluid produced by special glands at the base of the petals and/or the ovaries, collects around the ovaries in the cup like structures formed by the petals.  The quantity of nectar produced in each flower varies from species to species, about a teaspoon in tulip tree flowers (Liriodendron tulipifera) to very minute quantities as in the flowers of white clover (Trifolium repens). Nectar rich flowers are often called ‘honey flowers’.  Honey bees collect nectar, fill the cells of the comb with it and plug the cells with compacted pollen mass.  Nectar is dehydrated and converted into honey by enzymes.  Honey is the major source of energy for both the brood and the adults.

Pollen

Bee colonies would survive on nectar/honey or even on sugar syrup, but cannot lay eggs without pollen in the diet.   On the surface of the pollen there is a sticky layer known as the pollen coat, tryphine or pollen kit, which is a rich source of proteins, lipids, vitamins, phenolic compounds and minerals for the bees.  The extent and chemistry of pollen kit varies from species to species and the bees prefer pollen rich in pollen kit.

Flowers of many plants produce copious amounts of pollen, but the quantities vary very widely, from species to species.  The whole pollen component of a flower may not be available to bees.  During floral visits, worker bees pack pollen in concavities on the hind limbs, called the ‘pollen baskets’.  Because of the pollen kit, pollen stick to each other and also the different body parts of bees.  When the pollen kit is poor (as in the grasses), the pollen tend to be dry and powdery, and drop off the vector’s body.

Once at the hive, worker bees pack the pollen, along with resin from plants, into the honey comb.  Pollen germination, bacterial growth, anaerobic metabolism and fermentation of pollen in the comb are prevented by several chemical compounds including enzymes the bees add to preserve the stored pollen.  The processed pollen comb is called ‘bee bread’, later consumed both by the larvae and adult bees.

Royal jelly is secreted by the hypopharyngeal glands in the heads of young worker bees and is not related to the flowers the bees visit.  Rich in nutrients, royal jelly is used along with other diet, in feeding all the larvae in the colony, including those destined to become workers, but not the adults.

Bee colonies are perennial, though the life span of individual bees is about two or three weeks.  Bee colony populations are sustained through overlapping batch hatchings.

Bee foraging areas and Distances

The primary bee foraging areas are the various wild or cultivated plants in the vicinity of the hives.  Crop plant flowering being seasonal, bees depend heavily upon wild species of plants.  Honeybees also visit a number of species of wind-pollinated plants which do not contain nectar, such as the willow, oak, some grasses, but only to collect pollen.

Bee foraging ranges seem to have a relationship to the body size, particularly body the length of the bee species, the smaller bodied going farther.

Studies on tagged bees indicate that, in general, the foraging distances range from 50 meters to two km from the apiary, occasionally to seven or eight km, and exceptionally nine to 10 km.  Maximum foraging distances between nesting site and food mostly vary between 150 and 600 m, for 16 bee species studied.

Food Preferences of Bees

In general, bees forage on flowers of any species in the vicinity of the hive, on the basis of abundance of nectar and/or pollen and aggregation of flowers meaning fewer visits.  However, there seem to be a few other criteria of food preferences.

Genetic differences among the bee populations seem to influence species preference, as for example more to apple pollen than to other pollen.

Bees prefer pollen rich in pollen kit.  Bees seem to have preferred oil seed rape pollen containing greater proportion essential amino acids, than field bean pollen, suggesting that bee food preferences may depend upon the nutritional quality, probably operating thorough chemical signalling and experience.  Bees have also shown preference to certain food odors, also learnt by experience.

Bees and Palynology

In general, Palynology is the scientific study of pollen and spores of all plant species, but normally the term refers to the study of pollen.  Bees, nectar and honey are studied for pollen composition from three different perspectives: a) the external body parts of the bees to identify the species of plants the bees had visited, basing on the pollen in the pollen baskets and on different parts of the bee body, b) bees captured immediately after floral visits and dissected to analyze the pollen in the stomachs to identify the plant sources of nectar sucked in, and c) analyze honey for pollen for characterization of honey.

Bees do not visit every species in flowering in the vicinity of a hive.  Besides, the flowers of several species are not morphologically favorable for nectar or pollen collection by honeybees, particularly those with very small flowers or those with long narrow tubular corollas, which the bees cannot enter.

Bees only pack pollen in pollen baskets and do not consume raw pollen per se, from flowers they visit.  Since the floral nectar sources are usually close to the dehiscing anthers, some pollen from the anthers and pollen of other flowers on the bee’s body, fall into the nectar gathered by the bees.  A small component of pollen, which does not originate from flowers the bees had visited, is also found in the bee hives and honey.  Such pollen come in through wind, rain or accidental fall out.  Hence, pollen analysis of bees and hives should be carefully executed taking several factors into consideration, in order to be reliable.

The bee sucks nectar through a slender tube that enlarges into a thin-walled distensible sac called the ‘honey stomach’. Once in the honey stomach, the nectar flows over a regulatory apparatus (the proventriculus), that filters and controls the entry of food into the bee’s stomach. The nectar in the honey stomach is drawn back and forth into the proventriculus to remove debris such as pollen grains, fungal spores and dust.  About 90 per cent of pollen sucked into a bee’s honey stomach along with nectar are filtered out within 10 to 15 minutes after the floral visit.

The posterior end of the proventriculus extends into the mid gut (ventriculus), where food digestion and nutrient absorption take place.  A valve prevents the filtered nectar from entering the bee’s digestive system. However, this same valve will later allow pollen and debris removed from the nectar to pass into the bee’s intestines, and retained in the rectum until excreted.

The ‘yellow rain’ that often alarms people is the result of rapid defecation of massive quantities of pollen, by swarms of bees retuning to the hive, leaving thousands of tiny yellow spots all over the place.

Bees are captured immediately after floral visits and dissected to analyze the pollen in the stomachs to identify the plant sources of nectar.  An average of 7,100 pollen grains per ml of fluid were found in the honey stomachs of 38 bees captured and dissected immediately after each had completed feeding on the nectar of rabbit brush (Chrysothamnus nauseosus).   On the other hand, 30 honeybees that fed on orange blossoms (Citrus sinensis) and 32 bees fed on cotton flowers (Gossypium hirsutum) did not contain one single pollen grain of the respective species.

The analysis of honey for pollen (Melissopalynology or Mellittopalynology), facilitates construction of ‘pollen spectra’, the guides to qualitative and quantitative pollen composition of honeys, which are a potential basis to determine the quality and geographical origin of honey, the species of plants whose flowers the bees had visited and so the nectar sources.

Bees collect pollen and nectar from all sources in the environment and do not normally have any special preference to a particular species of plants.  Pollen of melon, cucumber, rapeseed, polygonum, alfalfa, clover, mint, thyme, sage, blue bells, thistle, white acacia, fireweed, eucalyptus, chestnut, basswood, orange blossom, buckwheat, and of several common weeds in the apiary environment were found in honey samples, in different parts of the world.

The quantity of pollen in a honey sample is not always directly proportional to quantity of nectar collected from a particular source.  In the case fireweed (Epilobium angustifolium), that contributed to 95 per cent of nectar, the pollen component in the nectar was only 6.3 per cent.  Rape seed (Brassica rapa) pollen were found to be abundant in a sample of honey but nectar contribution of this species was only 2 per cent.  With 28.3 per cent of white clover (Trifolium repens) pollen, nectar contribution of this species was less than two per cent.

The number of pollen grains in honey varies from 200 fireweed pollen grains per ml of honey to 41,000 white clover pollen per ml.  Unifloral honeys, such as melon, clover, cotton, canola, citrus or apple honey, are produced by hives located in the respective crop fields or orchards.  They contain more than 45 per cent of pollen from one predominant species, implying that most nectar was sourced by the bees from that particular species.  Special therapeutic and/or nutritional properties are attributed to unifloral honeys which command higher market prices.

C Kameswara Rao
Foundation for Biotechnology Awareness and Education, Bangalore, India
pbtkrao@gmail.com

Pollination

Pollination is the transference of pollen from the anthers (male structures) of a flower to the stigma (the receptive part of the female structures) of the same or another flower, mediated by abiotic or biotic means.  Pollination is the first in a series of crucial events that lead to seed and fruit formation.

Simple physical deposition upon agitation of the anthers/flowers, wind action and dew or rain constitute the abiotic means of pollination in the majority of wild and cultivated species of plants.  Several different biotic vectors such as insects (honey bees, bumble bees, butterflies or other insects) and small animals (bats, hummingbirds) render invaluable service by pollinating certain species of wild and cultivated plants.  Nevertheless, pollination by biotic vectors is rarely related to the species of the vector or the plants.  Even casual and incidental visitors like thrips, ants and predatory spiders are known to cause pollen transfer.    The pollen stick to each other and the body parts of insect visitors because of a sticky coating on the pollen surface, the pollen kit.  There is no biology involved even when biotic vectors carry out pollination.  They merely physically transfer pollen to another flower, which they visit next, whatever species that might be.

Biotic pollen vectors do not always pollinate the flowers they visit.  They may merely consume nectar, pollen or even some parts of the flower, without effecting pollination.
Bats were thought to be the pollinators of the West African scarlet bell (Spathodea campanulata), now a common avenue tree in the tropics, but it was found that the bats make a hole at the base of corolla, suck the nectar without ever touching the anthers or the stigmas, leaving the species to self-pollinate.   In the large cardamom (Amomum subulatum), honeybees take most of the pollen without pollinating and in the process deny feed to bumble bees, the actual pollinators, often seriously affecting crop yield.

In general, pollination, whether by abiotic or biotic vectors, is non-species specific, incidental or even accidental.

While in a vast number of species pollination can occur through both abiotic and biotic vectors, pollen of some species are accessible only to some insect vectors.  Flowers that are very small and those with long narrow tubular corollas are not accessible to bees, but are to butterflies and thrips.  In some species pollination does not occur in the absence of a specific species of the vector in the environment, resulting in reproductive failure.  Some famous examples of extreme vector dependence occur among the orchids such as the bee orchid (Ophrys apifera),  fly orchid (Ophrys insectifera) and  spider orchids (species of Caladenia), where the flower has evolved to resemble the female of the vector species to attract the males.  However, such cases are rare and occur almost always among the wild species.

Like wind, many insect pollinators only physically disturb the anthers, pollen and stigmas.   The bumble bees and hummingbirds agitate the flowers/anthers by a process similar to ‘sonication’ (buzz pollination) which displaces pollen from their anthers.

The pollen of many species are easily carried away by wind or animal vectors when the anthers are exposed and deposited on the stigma of any species, where stigmas are exposed.  Consequently, the stigma of a flower usually receives pollen of most similarly oriented species in its environment.

When the pollen of a flower are deposited on the stigma of the same flower, it is self-pollination.   When pollen are deposited on the stigma of another flower of the same species, it is cross-pollination.  There are many examples such as the pea where intricate floral structures have evolved to facilitate cross-pollination, though self-pollination occurs in such species too.  In several species self-pollination occurs even before the flower opens and the cross-pollination that occurs subsequently has no consequence.  In general, in most species either self- or cross-pollination can occur, ensuring seed and fruit set by one or the other means.

Pollen germination and viability

Pollen germination and pollen viability are different aspects.

The pollen of several different species in the vicinity of a plant are likely to land up on the stigmas of its flowers.  Pollen become dehydrated prior to transit and may be further dehydrated during flight, depending upon the temperature, relative humidity and the time in transit.  Rehydration of pollen upon landing on the stigma is the first crucial step in pollen germination, the process of production of long narrow pollen tubes.  The pollen kit contains proteins including lectins which play an active role in pollen-stigma recognition and pollen germination.    The pollen taken into the vector’s mouth do not germinate because the chemistry of the pollen kit is altered by the regurgitated contents of the mouth.

Viability is the further rapid growth of the pollen tubes carrying the male gametes, through the tissues of the stigmas and the styles, a long way to reach the ovules in the ovary.  This is a physiological process controlled by a number of physico-chemical factors.  Pollen inappropriate to the species/variety may also germinate, but the pollen tubes would not be capable of growing through the ovarian tissues due to factors that determine compatibility.  Additionally, there is a time factor that limits pollen viability and/or stigma receptivity.

Pollen of a very large number of species contain two nuclei at the time of dispersal.   One of these nuclei divides to form two male gametes by about the time the pollen tube reaches the ovary.  In several other species such as those of the grasses (cereal crops included) the pollen contain three nuclei, as the male gametes are already formed by the time of dispersal.  Pollen of such species have notoriously short viability, less than 10 minutes in rice to about two hours in some others.

Fertilization

The pollen tubes carry the male gametes to the egg cells in the ovules.  Fertilization, the fusion of the male and female elements, leads to embryo development and seed and fruit set.

When the egg cells of a flower are fertilized by the male cells from the pollen of the same flower, it is self-fertilization and in other cases it is cross-fertilization.  Genetically determined self-incompatibility is one means of ensuring cross-fertilization which facilitates new gene combinations paving way for further evolution of the species.  However, this has been an impediment in breeding such crops as mustards.

Pollination and fertilization in field crops

Species are reproductively isolated,  with the identity of species/varieties being maintained through several genetically controlled reproductive barriers that operate at one or more stages of pollen germination, viability, fertilization, embryo development and seed germination.  In the absence of such a natural isolation, there cannot be so many species and varieties of plants.  There is little chance of rampant natural interspecific hybridization.

Most field crop species are self-pollinated and self-fertilized, except those such as the cucurbits and corn, where the flowers are unisexual (contain either the anthers or the ovaries).  Several crop species such as the mustards, though self-incompatible in the wild ancestral states, are adapted to a high degree of self-fertilization on domestication. In a number of species like the pulse crops, self-pollination occurs even before the flower opens.  When self-pollination is possible, cross-pollination is largely inconsequential, as the former has an advantage of time, and even physiological competence.

What is actually important in crop reproductive biology is not whether there is self- or cross-pollination, but whether self-fertilization can occur and its genetic consequences.  This can only be determined by an analysis of the progeny for any visible marker characters or a genetic evaluation.

Most characters are controlled by two states (alleles) of one single gene, which may be identical (homozygous) or different (heterozygous) in a given individual.  Characters like growth and yield are simultaneously controlled by several genes each with two alleles (quantitative characters) where the inheritance is more complex.

Crop plants are selectively bred for beneficial characters through repeated crossing with one of the parents, which results in a high degree of homozygosity for the select characters.  Any heterozygosity for other characters is usually ignored.  Whether a crop is self-pollinated or cross-pollinated, is not an issue of serious consequence in most crops, because even when the pollen come from plants in another crop field, they are homozygous for the chosen traits, except when the traits in question are quantitative.