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.

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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.

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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.

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DNA banks or Gene banks are the ultimate facility for the ex situ conservationof genetic material to be sourced for a) genomic studies, b) to compile genomic libraries, and c) to isolate desired genes at will, for genetic engineering. However, currently they are not useful in regenerating whole plants, unlike the other forms of germplasm banking.

Any part of a plant yields DNA.  Plant material is dried using silica gel and stored at -800 C to extract DNA from it.  When fresh supplies not available, DNA can be obtained from dried plant material stored in botanical research institutions that house pressed and dried plant specimens called ‘herbarium’.  Though some fraction of the DNA from dried plants may be degraded, it would yield quality DNA in sufficient quantities.

DNA samples in the banks pass through extensive extraction procedures, minimizing cleaning process before using the sample.  However, the quality and concentration of DNA in a sample vary with species and so concentration procedures may be needed.  Even small quantities of DNA are adequate as they can be amplified a million fold using the technique of ‘polymerase chain reaction’ (PCR) that yields much larger quantities of DNA from minute quantities.

Samples in DNA banks are so well purified that they are stable at ambient temperatures for days in transit. While within the bank under controlled conditions, they are stable almost indefinitely. Ten-year old DNA samples were near perfect.

DNA banking is more economical than other forms of germplasm banking, as it occupies far lesser space, almost indefinitely viable and a small sample can be shared by many researchers through PCR amplification, without the need for repetitive extractions.

DNA BANKS

The following are some important examples among several DNA banks established in different parts of the world, which are networked for collaborative activity:

DNA Bank of the Royal Botanic Gardens, Kew

The DNA Bank at the Jodrell Laboratory, Royal Botanic Gardens, Kew, England,  contains over 22,000 samples of plant genomic DNA, all stored at -80°C. Information on the stored DNA is databased providing the names of the species, collectors, localities, etc. Each sample has a reference voucher of a herbarium specimen.  This database is linked to the Plant DNA C-values Database and the Database of the International Plant Names Index, which is quite useful.

DNA samples are provided to the collaborators all over world but can also be purchased by the others on a Material Transfer Agreement.

Plant DNA Bank in Korea (PDBK)

The PDBK website provides genomic lists of stored DNA and tissue samples, and their voucher information (label, specimen, and photo) held in PDBK and Korea University Herbarium (KUS), both located in the Graduate School of Biotechnology, Korea University, Seoul, Korea.

The Australian Plant DNA Bank (APDB)

The APDB located at Southern Cross University’s Centre for Plant Conservation Genetics at Lismore, is a comprehensive collection of DNA from both Australian native and important crop plant species. It also contains DNA of transgenic organisms developed through genetic engineering.  The APDB has invested heavily in advanced DNA storage facilities to ensure long term preservation of extracted DNA.

Missouri Botanical Garden’s DNA Bank (MBGDB)

Although the MBG describes its activity as ‘DNA Banking’, there is no evidence on its website that MBG banks extracted DNA samples.  It is a collection of samples of plant material stored at -20°C, suitable for DNA extraction.  Voucher specimens for these samples are deposited at the MBG or other institutions.  The material is provided to researchers against an agreement for molecular studies but not for commercial purposes such as bioprospecting, or screening for genes of interest in agricultural research.

DNA Bank Brazilian Flora Species

The Bank at the Rio de Janeiro Botanical Garden aims to preserve representative genetic material of the Brazilian flora, for plant conservation and biotechnology.

DNA Bank at Kirstenbosch

The Leslie Hill Molecular Systematics Laboratory at Kirstenbosch, in collaboration with the Royal Botanic Gardens, Kew, established a DNA bank to house genetic material of South African plants.  Extracted nuclear, mitochondrial and plastid DNA is stored at

-80ºC.  Each accession has a corresponding herbarium voucher.

NIAS DNA Bank

The NIAS, Ibanaki, Japan, is maintaining DNA samples and information gathered as a part of the genome projects of the Ministry of Agriculture, Forestry and Fisheries such as the Rice Genome Research Program (RGP).  cDNA clones, RFLP and other markers, PAC/BAC clones and YAC filters are available for distribution.

NATIONAL BUREAU OF PLANT GENETIC RESOURCES (NBPGR)

The NBPGR, New Delhi, India, has DNA fingerprinted about 2,200 crop varieties, and has expanding facilities for DNA banking.

GENE BANKS IN THE PRIVATE SECTOR

The private sector organizations, mostly multinational corporations, engaged in genetic engineering of crop plants have extensive collections of crop plant DNA.  Their extensive genomic libraries are an important source of useful genes for crop improvement.  However, this valuable material and information are not generally accessible to the public sector.

RESPONSIBILITIES OF DNA BANKS

The DNA banks have serious responsibilities in order to fulfill their mandates.  The more important of them are:

1. • Ensuring the authenticity of the scientific identity, source and geneology of the source species;
   • Adopting state of the art procedures of collection, recording, processing and preservation of DNA;
   • Maintaining quality DNA in adequate quantities;
   • Ensuring responsible use of the material supplied to others, assuring equitable benefit sharing by all parties;
   • Networking internationally, with other DNA banks, facilitating exchange of knowledge and material and to prevent duplication of efforts; and
   • Updating websites frequently and fulfilling the promises made.

RELATED ARTICLES:

CONSERVATION OF PLANT GENETIC RESOURCES
CONSERVATION OF PLANT SPECIES IN SEED BANKS
CONSERVATION OF PLANT SPECIES IN GERMPLASM BANKS

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Tissue Culture and Micropropagation

Cells differentiated through natural life processes in several different specific ways form the tissues and organs in organisms.  The earlier undifferentiated cells can be made to develop along specific directed lines to develop into tissues, organs and even whole organisms in synthetic media in the laboratory, through process a called tissue culture.   While natural cells, tissues and organs can be made to respond in tissue culture, the technique can also be used to modify natural or tissue culture originated cells, tissues and organs in almost any manner.  On the medical side the stem cells are being explored and exploited to develop tissues and organs for transplantation to replace defunct parts.

The undifferentiated cells in the growing points of plants and animals are totipotent in the sense they can be made to develop into any type of cell of the organism and so the whole organism, through appropriate procedures in the laboratory.  Elegant tissue culture protocols are widely used in a variety of experimental and production phases both for plants and animals.  Since all this happens in the laboratory glassware, it is called in vitro (in glass), in contrast to in vivo that happens in the living systems.

In plants the growing points that contain undifferentiated are called meristems.  The meristematic cells can be tissue cultured to produce tissues, organs and whole plants.  Tissue culture produced tissues, organs and plantlets, that constitute germplasm, can be cryopreserved regenerated at a later date when needed using tissue culture protocols.  Pollen can be cultured to produce haploid plants (containing only one set of chromosomes instead of the usual two sets) and even fertilization and seed development can be achieved in vitro.  This facility provides for the conservation of plant germplasm in germplasm banks, variously called in vitro banks or tissue banks or gene banks.

When a piece of meristematic plant tissue (explant) of any part of a plant is subjected to culture protocols, it develops into an undifferentiated mass of cells (callus), from which numerous heart shaped structures (embryoids) arise.  Each one of these embryoids can be cultured into a whole plant in the laboratory.  The embryoids encapsulated in calcium alginate or other suitable material, called synthetic or artificial ‘seeds’, can be cryopreserved for long periods till required for use.

As a very large number of embryoids develop from cultured callus, it is possible to produce thousands of plantlets.  The plantlets are transferred to soil to acclimatize (hardened) and taken to the field to raise a crop.  This process is micropropagation, a way of cloning, which is used extensively to produce genetically uniform plants.  These plants are disease free at least till they are taken to the soil.   There are hundreds of success stories of mass cultivation of plants produced through micropropagation with such crops as banana, plantation tree species as eucalyptus and poplars, and ornamental species as orchids.

Cell and tissue culture techniques are also largely required in developing whole plants from transformed cells in genetic engineering.

In vitro Banks require expensive infrastructure and expertise in collecting, processing and culturing plant material and to preserve and regenerate the cultured material into plants.  The viability of the banked germplasm varies with the experimental finesse and the species, and hence it has to be periodically checked.  Germplasm banking is more reassuring than seed banks, where once the seed viability is lost, the collections become unusable.  Like the seeds in seed banks, banked in vitro germplasm can be a source of DNA, for use in genetic engineering even when it is unviable.

Thousands of institutions have been engaged in plant tissue culture for over three decades and several succeeded in culturing a very large number of economically plant species, but only a few worked on crop plants.  While many institutions claim in vitro banks, only a few such as the Plant DNA Bank in Korea, are credible.   Some like the National Bureau of Plant Genetic Resources, New Delhi, India, bank only explant material.  Seed in the seed banks can also be used for tissue culture purposes.

Material in the in vitro banks suffers from some disadvantages.  Being much more sensitive to storage conditions, particularly temperature and humidity, than seeds and extracted DNA, the material requires very precisely controlled facilities to ensure longer viability periods.  Though plantlets produced through micropropagation can be transported rather easily, other kinds of in vitro banked material require special facilities for transit.  Tissue culturing requires high level of technical expertise and production costs are very high. 

Responsibilities of Germplasm Banks

The in vitro banks have serious responsibilities in order to fulfill their mandates.  The more important of them are:

1. • Ensuring the authenticity of the scientific identity, source and geneology of the explant source species;
   • Adopting state of the art procedures of collection, recording, processing, culturing  and preserving the cultured material;
   • Maintaining viability of the banked material and replenishing material of doubtful quality;
   • Ensuring responsible use of the material supplied to others, assuring equitable benefit sharing by all parties;
   • Networking internationally, with other in vitro banks, facilitating exchange of knowledge and material and to prevent duplication of efforts; and
   • Updating websites frequently and fulfilling the promises made.

RELATED ARTICLES:
CONSERVATION OF PLANT GENETIC RESOURCES
CONSERVATION OF PLANT SPECIES IN SEED BANKS
CONSERVATION OF PLANT SPECIES IN DNA BANKS

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There is a pressing need is for the conservation of crop genetic resources, but it is largely impractical to conserve the very large number of crop species and their wild relatives in their natural habitats.  A viable alternative is to conserve whole seed in Seed Banks.  The seeds are germinated to raise plants from them for use in crop improvement.

The important role seed banks play in the conservation of crop genetic resources is now globally recognized.

Various techniques have been developed for preserving seed, retaining their viability for longer periods.  As temperature and humidity are very critical factors, cleaned seeds are stored at around -20o C, often using silica gel in the seed containers to reduce humidity.  Seeds may also be stored over liquid nitrogen around -186oC (cryopreservation), which maintains retains seed viability for very long periods.

Seeds in the seed banks need to be protected from pests and pathogens while in storage, but the risk from seed borne pests and pathogens persists.

THE INTERNATIONAL SEED TREATY (IST)

The International Treaty on Plant Genetic Resources for Food and Agriculture of the United Nations (the International Seed Treaty, IST), in force since June 2004, is a comprehensive international agreement in harmony with Convention on Biological Diversity. IST aims at guaranteeing food security through the conservation, exchange and sustainable use of the world’s plant genetic resources for food and agriculture.  It also ensures a fair and equitable benefit sharing arising from such use. Further, it recognizes Farmers’ Rights to a) free access to genetic resources, unrestricted by intellectual property rights; b) to be involved in relevant policy discussions and decision making; and c) to use, save, sell and exchange seeds, subject to national laws.

SEED BANKS

Seed banks can provide controlled plant material of high quality and genetic diversity for research, eliminating the need for expensive expeditions.

There are several seed banks in different countries, at the national, regional and local levels.  Some of the best seed banks in the world are in Peru, Colombia, Syria, India, Ethiopia, and the Philippines. Most botanical gardens also have seed collections.

A number of other seed banks, some of them called ‘community seed banks’ have been operating for decades with high decibel propaganda.  These have been amassing seed collections in a haphazard manner and without any semblance of science in matters of collection, characterization, identification and preservation.  With the evaporation of the initial enthusiasm or motivating factors, the collections are forgotten.  Such hodgepodge attempts are resources for lobbyists and hobbyists to gain political mileage, rather than tools to promote conservation.

To be of any use serving the objectives of conservation, seed banks with state of the art storage facilities should be established at the national and international levels  and all such banks should be networked so that material, knowledge and expertise on  particular crops is available on a global scale.

The following are among the important international seed banking facilities:

a) The National Bureau of Plant Genetic Resources (NBGPR), New Delhi, India: NBGPR has over 3.43 lakh samples of 2.47 lakh varieties of 1,256 species, which include about 28,000 accessions of wild relatives of various crops.

b) Seed Banks of Global Network of Agricultural Research Institutions:

Ten international agricultural research institutions, co-ordinated by the Consultative Group on International Agricultural Research (CGIAR), Washington, are focused on crops and have extensive seed collections for such crops as rice, maize, wheat, barley, millets, pulses, oil seeds, tuber crops, banana, tropical forage and fruits.  The collections in these seed banks are well documented and the institutions are networked among themselves and with several other institutions.

c) The Millennium Seed Bank Project:

The Millennium Seed Bank Project (MSBP) at the Royal Botanic Garden, Kew, England, is one of the largest conservation projects.  MSBP’s 47 partner organizations in 17 countries intend to store 25 per cent of the world’s plant species by 2020. The Seed Information Database (SID) at Kew is an ongoing compilation of seed characteristics and traits world wide, targeted at >24,000 species.

d) The Svalbard Global Seed Vault:

0n February 26, 2008, the Svalbard Global Seed Vault (SGSV) opened near Longyearbyen (Norway), 600 miles from the North Pole.  SGSV is designed to hold 4.5 billion batches of seeds of the world’s main crops.

The SGSV is a glazed cave-like structure, drilled 500 ft below permafrost, in the middle of a frozen Arctic mountain topped with snow, with the goal to store and protect samples from every seed collection in the world, which will stay frozen.   An automated digital monitoring system controls temperature and humidity and provides high security.

The SGSV is an insurance against natural disasters such as earthquakes and tsunamis, or deliberate attacks like bomb blasts or human errors such as nuclear disasters or failure of refrigeration that may erase the seeds of any important species in the other seed banks or in the wild, in the other countries.  Such seed can be re-established using seeds from SGSV.

LONGEVITY OF SEED IN SEED BANKS

Any seed can imbibe water and swell, which is a mere physical process.  It may even germinate, and produce a short root, but longevity, the potential to develop into a plant is the most crucial factor in seed banking.

The claims that 10,000 year old seed of sacred lotus, arctic lupine and date palm germinated and produced plants were challenged.  Systematic scientific dating of seed and production of plants from them has shown that the germinated date palm seed is about 2,000 yr old and the lotus is 1,200 yr old.  The other authenticated reports on seed longevity are Canna (600 yr), and species of Liparia, Leucospermum and Acacia (200 yr).   Samples of 110 yr old cereal and weed seeds, stored in sealed glass vials in Vienna, germinated.  These are very exceptional examples of long seed viability.

Contemporary data indicate that willow seeds are viable for only a week.  The seeds of tropical rain forest trees have low viability.  Seeds of sugarcane, tea, and coco palm, have a life-span of up to a year. Rush seed was viable for seven years.

Data gathered from 13 worldwide seed-storage stations indicate that seeds of crops such as barley, corn, oats, potato, rice, soybean, and wheat, have half-lives between 3–13 yr, which means that in the period specific for each crop, seed viability comes down by 50 per cent.  Seeds with hard seed coats such as beans and soybean would be viable much longer than the cereals.

Seed banks should periodically check for pests and pathogens and test for seed viability, collect fresh samples from the plants obtained by germinating the old deteriorating samples.  Seed longevity can be maximized only in scientifically managed seed banks.  If the seed loses its longevity, seed banks become seed musea, though the DNA from the seed can be used in genetic engineering of crops.

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All life that constitutes Biodiversity depends on plants, the most crucial components of ecosystems in which all microorganisms, animals and humans, live.  Direct threats to plant survival are a combination of habitat loss, aggressive alien species, over exploitation and climate change.  The fundamental causes of these threats are rooted more in uncontrolled population growth, short sighted policies, ignorance or greed.  The consequent socio-economic factors are difficult to control.

Loss of plants leads to worsening food insecurity, increasing vulnerability to disease, lower material wealth, deteriorating social relations and restricted freedom of choice and action.  Despite such deep reliance on plants, continued misuse of ecosystems has taken us to a crisis point.

Conservationof species, particularly the agriculturally important ones which have an impact on human well being, has now attained paramount importance, in our efforts to provide for the sustainable utilization of biological resources, by preventing further loss.

In plants and animals, the DNA is the genetic material that maintains the organism’s continuity from generation to generation.  While a genome is one set of all genes of an organism, a gene pool is all the genes in a population of that species.

The genetic material of many organisms is of immense value and needs to be preserved for the benefit of the future generations.  Plant genetic resources should be available for use in research and breeding in agriculture and forestry.  These are a) actual currently cultivated varieties (cultivars), b) once favoured but now discontinued old cultivars, c) locally developed and preferred varieties called landraces that are or were grown and c) wild relatives of crop species.

The cells (including pollen), tissues, organs, whole single or few celled organisms, seeds or other propagules that serve as a means of regenerating the whole organism, constitute the germplasm which is preserved by various means in controlled storage facilities called biobanks or germplasm banks.  These are variously called as seedbanks, in vitro banks, gene banks and DNA banks, depending upon the material that is conserved in them.    Currently, extracted and preserved DNA cannot be used to regenerate the whole organism, but any chosen gene can be isolated for use in genetic engineering.

Several pressing considerations necessitate biobanking, of which the following are more significant:

1. It is estimated that 60,000 to 1,00,000 plant species, with diverse economic uses,  are under threat of extinction and need to be protected.
2. About three-quarters of crop biodiversity has been lost in the last century.  Eighty per cent of maize varieties known in the 1930s in Mexico no longer exist and in the USA 94 per cent of varieties of peas are no longer grown.
3. During the past 50 years many high yielding and/or otherwise better varieties particularly those with higher pest and disease tolerance, have continuously replaced the once favoured cultivars and landraces.  .
4. The dropped varieties may contain genes affording advantages to future agriculture.  For example, the genes for resistance against the late blight fungus that caused the Irish potato famine in 1845 were taken from South American potato varieties, not in active cultivation.
5. Wild relatives of crop species may contain genes useful in crop improvement. The genes for resistance against red rot of sugarcane that causes heavy losses were introduced from a wild relative (Saccharum spontaneum).
6. The Centres of Origin of the species that gave rise to crops and the original Centres of Domestication of crop varieties are a reservoir of crop genetic resources, most of which have already been lost and the rest need to be conserved.
7. Economics encourage farmers to drop crops and many farms now grow just one or two crops, with very high efficiency.  But on account of genetic uniformity, these crops may become vulnerable to the changes in the habitat, and pests and diseases.  Conservation of crop genetic resources is an insurance against such risks to food security.
8. Farmers have long stopped conserving seed for any reason.

Germplasm banking is a system to conserve crop varieties and their wild relatives, protecting them from the vagaries of climate, politics and human error.

APPROACHES TO SPECIES CONSERVATION

There are two main approaches to conservation wild or cultivated species:

In situ conservation: Conservation in the natural habitats, where evolutionary progression continues.  Over a period of time and several generations, the species/variety may change its genetic and morphological composition and even the desired traits may be lost, if they do not have any advantage to the species in that environment.  While whole populations of wild plant species can be conserved in bioreserves, cultivated species have to be cultivated and so are not amenable for in situ conservation.

Ex situ conservation: Conservation away from the natural habitats, which requires appropriate techniques for long term preservation of the seed or other material in biobanks.   On account of removal from the natural habitat, there is cessation of evolutionary progression, but the desired genes would be preserved.  The seed and other propagules may lose their viability sooner or later, but tissue culture methods may help in the revival of the material, if preservation techniques are appropriate.  Genetic engineering techniques would help in the recovery of the desired genes and their use in developing transgenics of the same or another crop.  While small populations of wild plant species can be grown in botanic gardens, cultivated species offer problems.  Ex situ conservation requires continuous professional attention, elaborate infrastructure and heavy financial inputs.

Since each has some disadvantages and some special advantages, a judicious combination of both in situ and ex situ approaches is needed for successful conservation of species.

RELATED ARTICLES:
CONSERVATION OF PLANT SPECIES IN SEED BANKS
CONSERVATION OF PLANT SPECIES IN GERMPLASM BANKS
CONSERVATION OF PLANT SPECIES IN DNA BANKS

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Below is another article on Prince Charles’ comments on GM food. Charles has long been an advocate for organic, sustainable agriculture, so his latest comments don’t come as a surprise. Some scientists in England are asking him to engage in conversation about the issue, hopefully he takes them up on it.

Dr. C Kameswara Rao

If we want to feed the world, we must go GM
The Telegraph
13/08/2008

Prince Charles is right on many things. Modern architecture - much of it an eyesore. Standards in schools - woefully low. Protecting Britain’s landscape - a noble aim.

On genetically modified crops, however - the issue that he discusses with Jeff Randall in today’s Daily Telegraph - I fear he’s wrong.

I am not a scientist, but rummage around in the scientific research about GM and a clear picture emerges: if we want to reduce starvation and “feed the world”, as Sir Bob Geldof et al tell us every Christmas, we must go GM.

The argument in favour of GM crops begins with a simple one: the world is growing fast.

Read more… »

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Scientists in the United Kingdom are attacking Prince Charles for calling biotechnology “technology [that] would cause the biggest environmental disaster of all time and lead to no food in the future.” In an article that appeared in Thaindian News, scientists called him “ill-informed” and said “that he had completely misunderstood the benefits and risks of GM crops.”

Dr. C Kameswara Rao

Prince Charles condemned for blaming genetically modified crops for global warming
Thaindian News
August 14th, 2008

London, August 14 (ANI): Scientists have condemned Charles, the Prince of Wales, for blaming genetically modified (GM) crops for global warming, and have called him shockingly ill-informed and negative.

According to a report in the Times, the Prince of Wales had launched an attack on genetically modified crops after claiming that the technology would cause the biggest environmental disaster of all time and lead to no food in the future.

Though the government, plant scientists and industry are promoting GM crops as part of the solution to global food shortages, the Prince said that biotechnology had already proved itself a dangerous failure.

Why else do you think we are facing all these challenges, climate change and everything? he said.

The role of gigantic corporations in food production was leading humanity towards absolute disaster, driving small farmers off their land into unsustainable, unmanageable, degraded and dysfunctional conurbations of unimaginable awfulness, he added.

The Princes comments, in which he blamed GM food and modern agriculture for environmental and social problems such as climate change and food shortages, were described by leading scientists as shockingly ill-informed.

Read more… »

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Scientists at the Laboratory of Plant Breeding at Wageningen University in the Netherlands have developed a method to identify and isolate genes to make potatoes resistant to Phytophthora infestans, also know as potato blight. The new method allows scientists to target more than one gene with the resistance, leading to hopefully more success. Scientists in the UK and US are also helping on this project.

Dr. C Kameswara Rao

New method discovered to make potatoes resistant to Phytophthora
CheckBiotech.org
August 8, 2008

Dutch, British and American scientists have developed a method to more quickly identify and isolate genes that can be used to make potatoes resistant to Phytophthora infestans, the dreaded potato blight. With this method, multiple resistance genes from different species of potatoes can be isolated and possibly used simultaneously. This offers the prospect of achieving sustainable resistance against the pathogen because it is less capable of breaking the resistance of the potato when multiple genes are involved.

According to researchers at Wageningen University in the Netherlands, the Sainsbury Laboratory at the John Innes Centre in the UK and Ohio State University in the USA, the best strategy to make potatoes resistant to the stubborn fungal pathogen Phytophthora is to develop so-called broad spectrum resistance. In their article, published on 6 August in the journal PLoS One, they explained that the current methods to discover resistance genes are too slow. Moreover, because they often concern only a single gene, these methods do not lead to sustainable resistance because Phytophthora can break single-gene resistance relatively quickly and easily.

Interaction

The newly developed method is based on the interaction of genes of the pathogen and genes of the potato. The response of the potato involves resistance genes in the plant, and the response of P. infestans involves so-called avirulence genes. The avirulence gene produces proteins (effectors) that are recognised by the resistance gene proteins of the potato; an interaction then takes place. By using effectors (proteins that are secreted by Phytophthora into the plant after infection takes place), researchers can relatively quickly identify and isolate the genes that are crucial to the interaction. Because the pathogen (Phytophthora) cannot switch off these proteins, but produces them constantly, genes that can recognise these proteins can potentially serve as resistance genes.

Read more…

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