Biotechnological Applications of Plant Cell, Tissues and Organ Cultures
In recent years, plant protoplast, cell and tissue cultures have become an important tool for crop improvement, commercial production of natural compounds and many more in the development of forestry. Swaminathan (1987) emphasized the significance of biotechnological application of in vitro
cultured plant protoplasts/cells/tissues as below : (i)
Tissue culture applications in order to capitalize upon the totipotency of cells, (ii)
Cell and protoplast cultures coupled with DNA vectors to overcome problems caused by barriers to gene transfer through sexual means; (iii)
Culture of plant cells for the production of useful compounds, (iv)
Extension and increase of efficiency of biological nitrogen fixation, and (v)
Transfer of genes for nitrogen fixation ability to non-fixing species.
Due to playing with plant tissues in laboratory, this technique has been referred by some researchers as a 'botanical laser' whose numerous uses are yet to be fully understood. In Gurgaon (Chandigarh), the tissue culture laboratories (Euro-India Biotech Ltd. in collaboration with Haryana Agro-Industries Cooperation, and Kiwi Callus (NZ) Ltd. New Zealand) have been set up for (i)
micropropagation of plants, (ii)
development and production of transgenic plants and seeds, (iii)
development of genetically engineered plants, and (iv)
production of pure and virus free plants of rare species of high value. Various aspects for plant improvement through tissue culture technology is given below in Fig. 9.1.
Fig. 9.1. Plant improvement through tissue culture technology.
Kuchko (1985) obtained somatic hybrid of wild and cultivated potatoes (S. tuberosum and E. chacoense)
and succeeded in the induction of organogenesis. The somatic hybrid plant inherited many characters viz., intermediate leaf morphology, stomata, forms and color of tubers, prolonged flowering, large and fertile pollen grains, high yield, resistance against Y-virus.
The best example of the application of anther culture in crop breeding and improvement is the production of anther culture derived rice and wheat varieties in China. About 50 varieties in rice and 20 in wheat have been developed by using this technique (Hu, 1985). The advantages of anther culture as a tool in plant breeding are : (i)
the availability of rapid method of advancing heterozygous breeding lines to homozygosity, (ii)
getting gametoclonal variations, and (iii)
early expression of recessive genes as well as variants and new forms (Zapata and Torrizu, 1987). However, haploid plant materials available as protoplast, cell and tissue culture systems are currently being evaluated for the use in transfer of foreign genetic material to select plant species by protoplast fusion, transformation, transduction and organelle transfer (Collins, 1975).
T.Murashige of the U.S.A. for the first time gave the concept of artificial seeds at a Symposium in Belgium in 1977. Artificial (encapsulated) seeds are the somatic embryos covered with a protecting gel. These seeds are compared to the true seeds. In these seeds, the gel acts as seed coat and artificial endosperm providing nutrient as in true seeds (Fig. 9.2). Water soluble gels (hydrogel) must be used as the protective gel. Usually Na/Ca alginate (a product of brown algae) is selected for encapsulation purpose because it is less toxic to embryos and easy to handle.
Methods of production
Following are the steps for the production of encapsulated seeds:
- Induce pseudoembryos (artificial embryos) from cell suspension culture.
- Mix embryos well with 2 per cent Na- alginate,
- Drop the embryos in a bath of calcium salt e.g. solution of Ca (NO3)2 for 30 minutes. This results in very quick complex formation at surfaces due to exchange of ions i.e. Na+ and Ca+ Consequently, individual embryo is enclosed into a clear and hardened beads of about 4 mm,
- Sieve the bead through a nylon mesh ; Ca(NO3)2 solution can be recycled, and
- Test the growth vigor of beads by plating in sand or soil amended with pesticides.
Fig. 9.2. Diagram of an encapsulate seed of plant
Kitto and Janick (1985a) produced Citrus
embryos in vitro
and tested 8 compounds for their synthetic coating properties on embryos. Out of the chemicals tested, a polyethyleneoxide (polyox WSR-N75) revealed good encapsulating properties. It was selected for use in further research with in vitro
produced carrot embryos (Kitto and Janick, 1985b). Later on Polyox coated embryos were kept in dry condition and then allowed for germination at suitable conditions. The germination percentage of the encapsulated embryos was low. The uncoated embryos did not survive after dry treatment. Kitto and Janick (1985c) applied a number of presumptive hardening treatments viz., (i)
high inoculum density (0.8 embryo suspension per 25 ml medium), (ii)
12 per cent sucrose instead of 2 per cent (iii)
chilling at 4°C during the last three days of the embryo-induction phase, and (iv)
amending 1mm abscisic acid in the nutrient medium at the time of embryo induction. These treatments should be combined with polyox coating. All the treatments increased the survivability of the coated embryos. Similarly, production of artificial seeds by encapsulation of somatic embryos in Eucalyptus
sp. has been reported by Muralidharan and Mascarenhaus (1987).
Production of Disease Resistant Plants
Many plant species, which propagate vegetatively are systematically infected by virus, bacteria, fungi, and nematodes. Their inoculum is carried over several generations resulting in continued adverse effect of productivity and quality of crops. In order to ensure highest possible yield and quality, it is necessary to provide disease free stock plants to growers. Tissue culture techniques have solved the problem and minimized the time of biological testing. Unless large scale population of pure inoculum of test pathogens are available, it is difficult to peruse the establishment of pathogenecity and crop loss - assessment as it is done in field condition. Now it has become possible to carry out such experiments in laboratory within short span of time by using tissue culture technologies. Miller and Maxwell (1983) have discussed the following advantages for the study of .several aspects of host-pathogen interactions and responses:
Production of Virus-free Plants
- Ability to isolate host cells without wounding,
- Control of inoculum of pathogen and number of host cells,
- Ability to change the nature of host pathogen interaction by altering the constituent of growth medium,
- Presence of only one or a few major host cell types, and
- Easy to apply and remove materials e.g. labeled precursors from cultured cells.
About 10 per cent of viruses transmit through seeds. In some cases, they are confined to seed coat (e.g.
TMV) or internally seed-borne (in legumes).
Moreover, viruses result in great loss, for example, potato leaf roll virus or potato virus X can cause up to 95 per cent reduction in tuber yield and potato virus X between 5 and 75 per cent depending on virus strain and host cultivar.
Tissue culture technique can be utilized for the production of virus-free plants either through meristem culture or chemotherapy or selective chemotherapy of larger explants from donor plants or dormant propagules or a combination of the two. Above plants were made virus-free by meristem culture by 1983.
Sood and Palni (1992) got early flowering in tissue culture raised and virus tested Easter lily plant (Lilium longoflorum). They
have described various steps for production of virus-free plants of Easter lily (Fig. 9.3) : (i)
bulb scale segments from commercially available bulbs were tested positive (virus indexing); segments were subjected to hot water treatment (40°C for 24 h), (ii)
surface sterilized segments transferred to MS medium supplemented with 2.5 mg/1 kinetin and 0.5 mg/1 IAA and solidified agar; direct organogenesis (shoot bud formation) was observed, (iii)
explants transferred to MS medium supplemented with 6-benzylaminopurine (2.5 mg/l and a-naphthalene acetic acid (0.5 ppm) for extensive rooting, (iv)
rooted plants transferred after 6 weeks to pots without any growth regulators, (v)
the plantlets transferred to soil. Normal flowering was observed in transplanted plants within 6 weeks. Virus indexing was carried out at various stages as indicated in diagram. Only those plants were retained which tested negative.
Fig. 9.3. Outline for production of virus-free plants of Easter Lily (based on Sood and Palni, 1992).
Morel and Martin (1952) for the first time successfully obtained a virus free plant through shoot meristem culture. Later on, several virus-free plants were regenerated in vitro
such as Pisum sativum, Trifolium repens
sp., Dactyl is glomerata
(from mild mosaic mottle viruses) and Lolium multiflorum
(from ryegrass mosaic virus) (Hu and Wang, 1983).
In vitro Selection of Cell Lines for Disease Resistance
For the study of disease resistance in vitro,
one of the important considerations is the selection of suitable type of culture e.g.
callus tissue, suspension culture, isolated cells or protoplasts. However, callus cultures have been widely used for study of expression of race-specific and non-host resistance, and offer several advantages over suspension cultures, isolated cells or protoplasts. The advantages are : (i)
case of initiation and maintenance of tissue in culture, (ii)
ability to add inoculum (spores and zoospores, etc.) directly on callus surface so that the culture medium does not act as direct source of nutrients for the pathogen, (iii)
the ability to follow the progress of infection and colonization of callus tissue by the pathogen using histological/cytological methods, and (iv)
phytoalexin . accumulation can be determined in pathogen Challenge tissue and related to the extent of colonization.
Subclones regenerated from callus cultures have been found more resistant than the original material against eye spot (Helminthosporium sacchari),
downy mildew (Sclerospora sacchari
)and Fizi disease (virus). Rao and Palni (1992) have reviewed in vitro
selection of cell lines for diseases resistance in plants. Rao (1989) raised a cell line of bajra pearlmillet (Penisetum americanum)
which was resistant to downy mildew caused by Sclerospora graminicola
Fig. 9.4. In vitro
selection of a cell line of Bajra resistant to downy mildew.
Heavily infected cultures with developing plasmodia lost their capacity to differentiate and finally died. The occasional appearance of green embryos from necrotic tissues was interpreted as possible sign of resistance which is expressed in cultures. Similarly, induction of resistance against Alternaria solani
has been achieved. The toxins isolated from A. solani
have been used as stress factor to toxin resistant clones of potato which eventually confer resistance to the pathogens.
Shepard et al.
(1980) have found an increased resistance to leaf blight disease caused by Phytophthora infestans
and Alternaria solani
in clonal populations generated from mesophyll cell protoplasts of the potato cultivar Russet Burbank.
They solved the technical problems of regenerating plants from mesophyll protoplast and then compared a number of protoclones (as they called them). Some were more resistant than others to P. infestans
and some to A. solani.
The work of Shepard and coworkers marks a new era i.e.
the entry of genetic engineering into plant breeding for disease resistance.
These techniques can be extensively applied for mass rearing of nematodes in vitro
and screening of resistant breeding materials and nematicides (and fungicides for fungal pathogens).
As a result of host cell-pathogen interactions, protoplasts of plant tissues are damaged due to secretion of toxins and enzymes. Therefore, by in vitro
test in a small flask, millions of protoplasts can be screened for resistance. They are equivalent to thousands acres of growing plants in the field conditions. Protoplast fusion or in vitro
pollination can, however, produce hybrids between species and genera of plants where normal sexual procedures fail.
A number of investigations show that intact plant resistance to pathogens also manifest itself when their cultivated tissues, cells or protoplasts are treated with the respective toxins. Toxin resistant callus plants have also been produced. Carlson (1973) for the first time obtained disease resistant plant by treating the tissues to be cultivated with toxins, then by regeneration plants from the stable cell clones. Chlorosis resistant plants were regenerated after treating haploid tobacco cells with an analogue to a bacterial toxin. Disease resistant plants induced by this technique are corn, sugarcane, cloves, tobacco, potato, etc.
Behnke (1980) has shown that leaves of potato regenerants, produced from calli and selected for resistance to culture filtrate- of P. infestans,
exhibited greater resistance to this filtrate than the leaves of control. No complete filtrate resistance correlation was observed when both the filtrate resistant and control plants were infected with fungal spores. It is obvious that resistance gene, which is expressed in the intact plant, is also expressed in cultured tissue. However, several factors affect the expression of resistance in cultured plants e.g.
temperature, inoculum density, balanced phytochrome, etc. The expression of race specific resistance in tissue culture has also been demonstrated for potato in response to P. infestans.
It has been found that tissue culture aggregates from the variety majestic with no resistance (R) genes to P. infestans
stimulated growth of races of pathogen, whereas aggregates from the variety onion which contain R1
genes did not stimulate the growth (Ingram and Robertson, 1965). In addition to tomato / P. infestans
system, other systems in which expression of disease resistance in tissue cultures has been studied are, tomato (Lycopersicon esculentum)IP. infestans
sp. system, soybean (Glycine max)/ Phytophthora megasperma
system, tobacco/tobacco mosaic- virus system, etc.
Miller and Maxwell (1983) have described the several advantages of tissue culture systems over the suspension cultures, isolated cells or protoplasts for the study of expression of race specific host resistance. These are:
the ease of initiation and maintenance of cultured tissue,
the ability to add inoculum directly to callus so that the tissue culture medium would not be a direct source of nutrition for the pathogens, and
the ability to follow cytologically the progress of infection and colonization of callus tissue by the pathogens and the hosts response.
Production of Stress Resistant Plants
Biochemical mechanisms exist in cultured cells which determine the resistance to biocide chemicals and provide the theoretical promise for selection in vitro.
Zenk (1974) has reported cell suspension tolerant to 2, 4-D when the cells were subcultured for 6 months in liquid medium supplemented with increasing amounts of herbicides. The cells were able to grow in 1mM (milli mol) 2,4-D, while the control suspension was completely inhibited at 0.3 mM 2, 4-D. The mechanism of tolerance involves an enhancement in the metabolism of 2, 4-D. Widholm (1977) observed that carrot cells lost the tolerance to the herbicides when transferred to 2, 4-D free medium. It suggested that tolerance was the result of induction of enzymatic systems responsible for the degradation of 2, 4-D.
Chaleff and Persons (1978) reported for the first time that regenerated plants from stable cell lines were resistant to herbicides. They also obtained mutant tobacco plants with monogenetic dominant resistance to picloram. The promising possibility of selecting for herbicide resistance in vitro
was supported by many workers in many plants e.g. Citrus,
barley and tomato.
Uses of protoplasts as a system to select cell lines tolerant to herbicides have not been extensively explored. Protoplast technology can be used to increase the possibility to obtain monoclonal lines and offer the opportunity for intraspecific transfer of cytoplasmic factor of resistance to some type of herbicides. A potential application of transfer of herbicide tolerance factor is the transplant of cytoplasrnic organelles, for example, chloroplasts.
Transfer of nif gene to Eukaryotes
Nitrogen fixing ability, a genetic character, exists in prokaryotic diazotrophs. However, one of the major tasks is the transfer of this character to eukaryotes. In recent years, researches are being done to solve this problem through tissue culture techniques coupled with the recombinant DMA technology. Historically, nitrogen fixation by rhizobia was believed to occur through symbiosis. For the first time an excitement was caused in scientific community with the discovery by Holsten et. al
. (1971). They obtained active rhizobia in the absence of nodules, leghaemoglobin and bacteroids which were apparently necessary in the intact plants. They established Rhizobium japonicum
on cell suspension of soybean roots. Callus induced from root explants of soybean on a specific medium was inoculated with R. japonicum.
Later on it was microscopically observed that infection threads were formed by the bacteria which were present between intracellular spaces. They multiplied inside cells. Moreover, development of nitrogenase in soybean callus - Rhizobium
system growing on solid medium was also observed by Child and La Rue (1974). It was found that only specific (isomorphic) cells of callus are vulnerable to infection by the bacteria.
In addition to improvement in the bacterial strains and increased nodulation, it is necessary to seek those genotypes with the efficient photosynthesis and improved partitioning of carbohydrates to nodules.
The real challenge lies in achieving greater input of biologically fixed nitrogen into nonlegume crops. Improvement of associative N2
fixation by sugarcane, wheat and the crops associated with Azospirillum
will have the same objective as Rhizobium
works (Ruschel and Vose, 1983).
In addition to work done successfully: on m/gene transfer, there are other important genes which have been clones, for example, (i)
phaseolin and leghaemoglobin genes of soybean, (ii)
storage protein genes in soybean, (iii)
genes of ribulose bisphosphate carboxylase/oxygenase (RuBP case) of pea, maize, wheat etc. Success achieved on these aspects would certainly promote in green revolution.
Moreover, improvement in primary productivity by conversion of C3
plants into C4
ones through genetic engineering techniques hopefully would increase the primary productivity. It is well established fact that photorespiration lowers the capacity of C3
plants in temperate zones which results in the inhibition of net biomass of about 50 per cent. This is caused by RuBPcase due to its reaction with carbon dioxide and oxygen. The same enzyme leads the reaction of both carboxylation and oxygenation. When the concentration of carbon dioxide is low and that of oxygen is high, the later competes with the former and leads oxygenation by RuBP oxygenase with the production of glycolic acid induced by photorespiration rather than producing phosphoglyceric acid in a normal way through carboxylation by RuBP carboxylase.
By genetic engineering techniques, if the gene encoding RuBPcase is modified which would combine only with carbon dioxide but not with oxygen, only then it would be possible to convert C3
plants into C4