In vitro Pollination & Fertilization

Reproductive isolation is considered to play a key part in evolution.Plants and animals have developed a range of strategies that minimize gene flow between species.In plants, these strategies involve either pre-zygotic barriers, such as differences in floral structure and pollen-stigma recognition (inhibition of pollen tube growth), or post-zygotic barriers (malformation of endosperm and the inhibition of germination) which are less well understood and affect seed development from fertilization to seed set.

Wide Hybridization

In most crop improvement programme often wide hybridization is resorted to transfer the genes for abiotic and biotic stress from alien genera. Involves interaverietal, inter-varietal, inter-genera and inter-family crosses to transfer the target genes from the donors.Eg. Wheat & Rye (Inter-genera)

Rice: Wide crosses of interaverietalMost failure in these crosses are due to

i) self incompatibility, ii) cross incompatibility etc.

In vitro pollination & Fertilization

To overcome the barrier of hindering the growth of the pollen grain on the stigma or style, a part of the stigma or style may be cut and the pollen grain may be placed on the cut surface of the ovary or transferred through a hole in the ovary wall called “intraovarian pollination”

Eg. Papaver somniferu, P. rhoeas, Argemone mexicana

Another approach to overcome the barrier to pollen tube growth is direct pollination of cultured ovules (in vitro ovular pollination) or excised ovules along with placenta (in vitro placental pollination)(Developed at University of Delhi by Maheswari and Kranta, 1954 to overcome the self incompatibility in Papaveraceae and Solanaceae)

In vitro pollination

Stigmatic pollination

Ovarian pollination

Placental pollination

Diagrammatic representation of the in vitro pollination

Other techniques (in vivo) to overcome prezygotic barriers are:

a. Bud pollinationb. Stump pollinationc. Heat treatment of the styled. Irradiation and e. Mixed pollination

Development of seed through in vitro pollination of exposed ovules is termed as “Test tube fertilization”Seed formation following stigmatic pollination is termed as “ in vitro pollination”In vitro fertilization (IVF) is a process whereby reproductive structures are isolated and introduced to each other enabling fusion of gametes to proceed under culture conditions.IVF has been accomplished by using isolated male and female gametes of maize (Kranz et al. 1991; Kranz and Lörz, 1993; Faure et al., 1994), wheat (Koväcs et al., 1995), and tobacco (Tianand Russell, 1997).

In vitro pollination

In vitro pollination: Methodology

Ovaries should contain large number of ovules (In solanaceae many members viz., Nicotianatabacum, N. alata, N. rustica and Petunia hybrida), and in Papavaraceae and cryophillaceae, the placenta are covered with several hundreds of ovules.Isolation of ovules with out any damage as possible in these spp. which contributing maximum to the success in the in vitro pollination.The other requirement is the pollen which should be viable and able to germinate.There must be abundant growth of pollen tubes all over the ovules and placenta in the culture.

Conditions required for successful IVF

Other requirements:

Before to start, the information on

1. Time of anthesis2. Time of dehiscence3. Time of germination of pollen tubes into

ovules4. Viability of ovules and fertilization inside

the embryo sacs etc. are essential for successful IVF.

Disinfection of materialsThe buds to be brought to the laboratory for aseptic culture just before the anthers are at the stage of dehiscenceThe whole pistil (after removing petals and sepals) or the ovaries alone are sterilized by a quick rinse of 70% alcohol. (Ovaries of plants grown in open air requires longer period of sterilization)The ovary wall should be carefully peeled with scalpel, needle to expose the mass of ovules attached to the placenta.To perform stigmatic pollination the excised pistils are to be carefully surface sterilizedwithout wetting the stigma.

Preparation of pollen & ovary for IVFAnthers collected form the bud are kept in as sterile Petri dish containing a pre-sterilized filterpaper until their dehiscence.Generally the pollen deposited directly on the cultured part of the pistil performs better than that spread on the medium around the ovules.In graminaceous family the ovaries are well protected by many layers of husks and hence the surface sterilization is not required.In maize husks are severed after 2-4 days of silking with a scalpel.Ovaries are removed and transferred to the medium in a Petri dish.

Isolation of dimorphic sperm cells of Nicotiana tabacum

a: Population of larger sperm cells, representing the Sua . b: Population of smaller sperm cells representing the Svn .

(Yang et al., 2005)

In vitro pollination

Pollination is done directly on the silks or the ovules in vitro.Twenty four hours after the pollination silks are clipped off and the Petri plates sealed.To avoid the contamination with bacteria a brief disinfection with 95% alcohol (a brief rinse of inner husks or 30 min. treatment with 1% Famosept (Sladky and Havel, 1976) may be necessary.

Culture of ovule & ovary after IVPThe growth of the pollen tube on the barren ovule is affected by the presence of moisture on the surface of the ovule.The ovules may be wiped with a filter paper and then covered with pollen grains.After 4-6 days the ovules contain single celled zygote which requires a complex growth condition.In self pollinated species, the ovules with zygotes are kept along with placenta until seed formation while in cross pollinated species they require the placenta only in the initial 6-8 days.Afterwards they can be transferred to a fresh medium without placentaOvule culture is proved useful in raising interspecific hybrids in Gossypium, Helianthus and Trifolium

Ovary Culture after PollinationNitsch (1951) developed the in vitro technique for the ovary culture and successfully cultured the ovaries of Cucumis and Lycopersicum.Addition of vitamin B to the medium resulted in the development of normal fruits and viable seedsEnrichment of medium with IAA and coconut milkinduced larger fruits than the fruits formed in in vivocondition (Kanta & Maheswari, 1963).The floral envelops (lemma & palea) play an important role in the development of fruit & embryo in monocots.Ovaries excised soon after the fertilization in Triticumaestivum & T. spelta develop in the culture only when the floret envelops remain intact.This requirement of floral envelop with the excised ovule in monocots for the fruit development is known as “Hull factor”.

Ovary Culture after Pollination contd…

In the absence of the hull factor, DNA synthesis and cell elongation in barley embryo cells takes place but cell division does not occur.Similarly the association of perianth in dicots has been found necessary for the development of fruits.Several interspecific and intergeneric hybrids were produced between sexually incompatible parents in the family, Cruciferae with the aid of ovary culture (Batra et al., 1970).

Factors affecting the seed set after IVP/IVF

1. Physiological status of the explant2. Culture medium3. Storage condition4. Genotype

Physiological status of the explantThe physiological state of the pistil at the time of excising the ovules or ovary highly influence the seed set after the IVP. Wetting the surface of the stigma (in stigmatic pollination) may leads to poor germination or bursting of the pollen tube followed by poor seed set.

Physiological status of the explant contd….Pollen germination & growth of pollen tube through the style may influence the synthesis of some proteinwhich may prevent the entry of pollen tube into the ovule.Hence it is necessary to find which part of the pistil, the barrier exists.To increase the success rate, it is necessary to excise the part which synthesis the inhibitory protein and to directly pollinating the remaining portion of the pistil under in vitro conditionThe time of excising the ovules from pistil has a definite influence on seed set after IVP.Ovules excised 1-2 DAA showed higher seed set than on the day of anthesis.In maize spikes pollinated 3-4 days after silking yields better results.

2. Culture mediumNitsch’s mineral salt and White’s vitamins were used along with 5% sucrose to culture the ovules successfully by Maheswari, (1958). (for the constituents refer Table 3.1 in pp. 118 of text book by Razdan).Addition of kinetin and CH (Casein

hydrolysate) was essential to promote the initial growth of the embryo.Several orchid ovules grew successfully in a simple 10% sucrose solution.But the ovules of Zephyranthes required coconut milk or casamino acid in the Nitsch’smedium.

2. Culture medium contd……In Trifolium ripens, ovules (1-2 DAP) required the supplementation of the medium with juice prepared from young fruits of cucumber or water melon.GA3 (10 Mg l-1) in addition to fruit juice further improved the seed development in the young ovulesSteward & Hsu, (1978) developed a medium for raising intraspecific & inter-specific hybrids from young ovulesPresence of 10 μg l-1 IAA or kinetin improve the number of seed per ovary.According to Genginbach (1985) the N source does not affect the fertilization frequency in the excised ovaries of maize, but the amino acids as the source of N is required for the optimal kernel development & growth.Osmolarity of the medium also affects the development of excised ovules.Normal sucrose 4-10%. But a ovule with zygote with few endosperm nuclei- 6%; ovules just after fertilization - 8% sucrose.

3. Storage conditionsNo precise information on the effect of temperature and light on the test tube fertilizationUsually the first step of the process occur at room temperature without special lighting.Later the ovaries are transferred to 22-26°C

4. GenotypePollen grains of crucifer are difficult to germinate and a modified technique is requiredDipping Brassica ovules in 1% CaCl2 before pollination followed by transfer to Nitsch’smedium is required to successful seed set.

Application of in vitro pollination

Four different major areas are:1. Overcoming self-incompatibility2. Overcoming cross-incompatibility3. Haploid production through parthenogenesis4. Production of stress-tolerant plants.5. The use of IVF for research into the cellular

and molecular control of fertilization in higher plants and its application as a tool in biotechnology

1. Overcoming self incompatibility thru IVP

Petunia axillaris and P. hybrida are self incompatible species.Barrier to the germination of pollen exists in the ovary.This was overcome by IVP and seed set has been reported. In Petunia the self incompatibility can also be overcome by bud pollinationSeveral interspecific, intergeneric and interfamilial crosses were attempted through in vitro ovular, placental pollination.

++ Abundant germination3: embryo formed

Germinating pollen tubes

Twin Embryo 3 d after in vitro pollination

Pollen grains of the gymnosperm species Ephedra distachya and Pinus wallichiana germinated abundantly on the invitro cultured placentae of the angiosperm species

Interspecific, intergeneric and interfamilial crosses attempted through IVP

3. Haploid production through in vitro pollination

Haploid production through in vitropollination in Mumulus luticus when pollinating its exposed ovules with Torenafounieri was reported.The haploid of M. luticus developed parthenogenetically which otherwise could not have been obtained through anther culture.Such parthenogenetic development of haploids also been reported in Hordeum vulgare, Nicotiana tabacum & Triticumaestivum

Haploid plantlet regeneration through gynogenesis in Citrus clementina cv. Nules, induced by in vitro pollination with pollen grains of Oroblanco, a triploid cultivar of grapefruit.It indicates that parthenogenesis induced in vitro by triploid pollen can be an alternative method to obtain haploids in monoembryoniccultivars of Citrus.Pollination and mature stage of pistils was necessary for gynogenic embryo regeneration.Fourteen haploid gynogenic embryos of Nulesclementine were obtained

Germination of a haploid embryoGynogenic embryos breaking through the

ovary

Germination of a haploid embryo

4 months old pollinated pistil

1 month old pollinated pistil

Stigma exudate and Oroblanco pollen grains

A haploid citrus plant obtained through in vitro pollination

Haploid production through IVP in wheatTo improve haploid plant production in durum wheat the haplo-method involving intergeneric crossing with maize followed by embryo rescue was used.The wheat genotype was significant for ovarydevelopment, embryo and plant formation, whereas the maize genotype was significant only for embryo formationThe significant effect of the wheat genotype on embryo formation was found.Effect of the rescue medium for embryos on plant yields showed MS/2 and B4 gave significantly higher percentages of plants

Haploid wheat embryo and its development after a durum wheat x maize cross

a: Embryo 2 DAP at the time of its rescue in culture (bar = 1.5mm)b: Germinated embryo after 2 week of culture (bar =1.5 mm)c: Haploid plant at the four leaf stage after about 5 weeks in a culture tube

Intergeneric hybridization between wheat (Triticum aestivum L.) and a wild weedy species, Imperata cylindrica (2n = 20) resulted in the recovery of a high frequency of wheat haploids, which were obtained through the elimination of I. cylindrica chromosomes.Comparisons based on the efficiency of I. cylindrica and maize (Zea mays) as pollen sources indicated that Imperata-mediated haploid production is equally efficient.

Culturing condition

The embryos obtained from wheat X I. cylindricacrosses were excised under aseptic conditions and cultured on a nutrient medium which comprised the standard MS medium, supplemented with 0.5 mg/l kinetin, 400 mg/l glutamine, 20 mg/l each of L-arginine, L-cysteineand L-leusine, 30 g/l sucrose and 8 g/l agar agar.The embryos obtained from crosses of wheat X I. cylindrica possessed a haploid set of wheat chromosomes because of elimination of I. cylindrica chromosomes (as revealed by the cytological study of the root tips of the haploids)

4. Production of stress-tolerant plants

Maize plants tolerant to heat can be produced through in vitro pollination at high temperature.Pollen grain at temperature 38ºC were able to effect fertilization and the resulting maize plants expressed the trait for heat tolerance.Additionally these plants exhibited increased vigour and grain yield.IVP induced haploids and doubled haploids are potential source for identifying plants tolerant to many abiotic stresses combining IVP & in vitro screening techniques.

Other applications of IVFDifficulties in isolating gametes of higher plants have impeded our understanding of gamete physiology, activation of development and early embryogenesis in flowering plants. However increasing number of tools now available to manipulate male and female gametes of higher plants provides numerous opportunities for scientific and biotechnological progress (Fig.).Isolated gametes can be analyzed directly during IVF with modern cellular and physiological probes, while means of regulating sexual reproductive development are being refined.

(Zygotic) Embryo culture TechniqueIn angiosperms, the embryo is the miniature sporophyte resulting from the fertilized egg or zygote and the endosperm is the main nutritivetissue for the embryo.These are the products of double fertilization during which out of the two male gametes, one fertilizes the egg to form zygote and other fuses with secondary nuclei to form triploid endosperm.The culture of embryo and endosperm has been practiced by plant breeders for over half a century.Among the two techniques, the former has a long list of success stories whereas the latter has very narrow success

Embryo cultureIn seed bearing plants, embryos are easily accessible.They can be separated with relative ease from the maternal tissues and cultured in vitro under aseptic conditions in media of known chemical composition.Hanning (1904) first reported his systematic attempt to culture isolated embryo of Cochleria and Raphanus (Cruciferae).He successfully raised seedling from the cultured embryo using semi-solid medium containing mineral salts & sugar.Later Laibach (1925 & 1929) cultured excised matured embryos from the seeds of an interspecific cross, Linum perenne X L. austrianum and succeeded in raising the hybrid

Types of Embryo cultureAccording to Pierik (1989) there are two types of embryo culture1. Culture of immature embryo:Mainly used to grow immature embryos of hybrids which fails to germinate which require a complex medium.Success of this culture mainly depends upon the developmental stage of the immature embryoBy culturing immature embryo, it was able to develop bulkier seeds than in in vivo condition as reported by Monnier (1980)

Types of Embryo culture contd…

2. Culture of mature embryoMature embryos are excised from the seeds and cultured mainly to avoid inhibition in the seed germination.This type of culture is relatively easier as embryo require a simple nutrient medium containing mineral salts, sugar and agar.

Embryo culture techniques1. DisinfectionEmbryos develop normally inside the ovules which in turn covered by ovariesSince they are already in a sterile environment they do not require disinfection unless they are injured or seriously infected.Entire ovules are just sterilized following the standard methods of surface sterilization and embryos are dissected out and transferred to culture medium under aseptic conditions. In orchids, seeds are minute and with highly reduced seed coat, the entire capsules are to be sterilized and the seeds are removed under aseptic condition.

Surface sterilization of materialBy immersing the material in hypochloritecontaining commercial bleach (5-10% Chlorox, 0.45% sodium or calcium hypochlorite) for 5-10 min.Or ethanol (70%) for 5 min.A small amount of (0.01-0.1%) of a surfactant(Tween-20, Tween-80,Teepol or Monnoxol) to the disinfection solution increases the tissue wettability.Magnetic stirring, ultrasonic vibrations or a vacuum applied during soaking of the plant material in the disinfectant solution will reduce the possibility of trapping the air bubble on the plant materials.

Excision of embryosExcision operation should be performed under aseptically in a laminar air flow hood.A stereomicroscope (90X) with a cool ray fluorescent lamp is required for excising smaller embryos.Common tools used are: Forceps, dissecting needles, scalpels, razor blades and Pasteur pipettesMature embryos can be excised easily by splitting open the seed.Excision of smaller embryos requires careful dissection under a dissection microscope esp. where the embryos are embedded in liquid endosperm. (see Appendix 11.1 &11.2 in pp 145, 146 of text book by Razdan)

Embryo-Endosperm transplantAbortion of embryos at early stage of development is due to the non availability of nutrients.Brink (1951) demonstrated the in vitro growth of immature embryos (300-400μm long) of Hordeum by surrounding them with embryos excised from another seed of the same species.Kruse (1974) implanted immature embryos of Hordeum X Secale on cultured Barley endosperm and succeeded in getting hybrids than the normal method of embryo culture.Generally endosperm older than the embryo by 5 days was more efficient as a nurse tissue for the growing embryo

Normal endospermHybrid embryo

Endosperm Transplant Technique (Williams & De Lautour (1980)

Successfully used in developing interspecific & intergeneric hybrids in forage legumes which do not grow in normal conditions

Culture Medium-Nutrition requirementThe requirement of culture medium depends on the types of embryo culture. They may be either post-germinal or pre-germinal. In the case of post-germinal embryo culture, embryos are cultured only to speed up the process after germination. This can be achieved with less complex medium or even with sucrose or glucose solution. In pre-germinal embryo culture, immature embryos are cultured to get plantlets, where the embryos require a complex nutrient medium. Includes modifications in the composition of mineral salts, organic nutrients and growth regulators

Nutrition requirement contd…..The composition of the culture medium has to be formulated to suit the developmental phase of the embryo.There are two phases in embryo development

(1) heterotrophic phase in which the embryo draws its nutrients from the endosperm and the surrounding maternal tissues and

(2) autotrophic phase in which the embryo is metabolically capable of synthesizing substances required for growth

Embryos excised at near maturity stage are completely autotrophic and grow on simple medium comprising salts with a carbohydrate source.Immature embryo which are heterotrophic requires complex medium including vitamins, plant extracts and plant growth regulators.

Monnier (1976) developed technique which allows complete development of Capsellaembryo (early globular stage) upto germination without moving them from the original position in the culture plate as below:

Mineral salts

Inorganic nutrients of MS, B5 and White’smedia with certain degree of modification are the most commonly used media for embryo culture.Monnier (1976) modified the MS medium composition for higher survival rate of cultured immature embryo.Contained high level of potassium (adding 350 mg l-1 KCl) and calcium (double concentration of CaCl2) and reduced levels (approx half) of ammonium (NH4NO3) and FeEDTA and double concentration of MS micronutrients.

Table contd…

CarbohydratesSucrose is the most commonly used source of energy for embryo cultureIn maize, addition of maltose, lactose, raffinose, or mannitol may be required.Superiority of Glucose over sucrose is reported in the embryo growth of Carex lucida, several spp of Rosaceae and Lilium hybrids.Glucose & sucrose besides nutrition, also maintain osmolarity of the culture medium.Mature embryo grow fairly well at low sucrose, but younger embryos require higher conc. of sucrose.Various concentrations of sucrose used for different species band age of embryo is given below:

Role of sucrose in the medium

Nitrogen & VitaminsEmbryos have enzyme system to reduce nitrates to ammoniumAmmonium nitrate is superior to sodium nitrate, potassium nitrate and ammonium diphosphatePresence of ammonium is essential for the growth & differentiation of embryosHanning (1904) reported that asparagineenhanced embryo growthGlutamine to be a superior source of N in Capsell sp.CH (a amino acid complex) widely used as an additive to embryo culture media.Optimum level of CH is 500 mg l-1.

Natural Plant ExtractsCoconut milk (CM) assumed to possess some “embryo factor” which presumably make up deficiencies of certain sugars, amino acids, growth hormones and other metabolites of the culture medium.CM stimulates the growth of young excised embryos of sugar cane, barley, tomato, carrot, interspecific hybrids of Vigna and fern spp.Water extracts of dates, bananas, hydrolysates of wheat gluten and tomato juice were reported to promote growth of embryos. Alcohol diffusates of young seeds of Daturaand Sechium had comparative effect of CM and Lupinus has got twice the effect of CM.

Growth RegulatorsAuxins & cytokinins are not generally used since they induce callus formation.At very low level (0.01 mg l-1), GA promotes embryogenesis of young barley embryos (without inducing precocious germination)ABA (abscisic acid) also has similar effects on barley and Phaseolus embryos

pH of the mediumEmbryos grow well in a medium with pH of 5-7.5Generally the medium pH is adjusted 0.5 above the desired pH to compensate the uncontrollable change during autoclaving process

Incubation conditions

Incubation temperature of 25 ±2°C normally supports the growth of majority of embryos.Sometimes the incubation temperature may vary with the genotypes of the same speciesZennia, Phaseolus and cotton adopted to warm temperature (27- 30°C) to culture embryos.Whereas in Brassica hybrids, rice, barley in cold regions or seasons require temperature of 17-22°C (Hu & Wang, 1986).

LightIt is advisable to incubate immature embryos of barley, flax, Aegilops x Hordeum hybrids and interspecific hybrids of Allium in darkness before transferring them to light for germination.A recalcitrant secondary dormancy is induced when these embryos are exposed to 4000 lx for more than 4 hrs of light during the initial four days.This suggests that initial incubation of embryos for 4 days is essential.

Role of suspensor in embryo cultureThe suspensor is an ephemeral structure found at the radicular end of the proembryo and attains maximum development by the time the embryo reaches the globular stage. Generally, it is difficult to excise the suspensor along with the embryo because of its small size and delicate structure. Older embryos (500 μm or more in length) appear to grow well with or without a suspensorObservations showed that attachment of the suspensor with young embryos of Phaseolus coccineus, or its placement in close proximity (when detached) of an embryo, on the culture medium strongly stimulated the development of embryos to maturity compared to those cultured without the suspensor. The requirement of the suspensor may be substituted by the addition of GA or ABA to the culture medium

Precocious GerminationPlant physiologists and biochemists conceive embryo development as a linear progression from zygote formation to germination. According to Walbot (1978), embryo development can be classified into five stages (Table 11.6).

Excised embryos on a nutrient medium tend to bypass the stage of dormancy.Directly develop into a weak seedling.This phenomenon of seedling formation without completing normal embryogenic development is called precocious germination.CH promotes further embryogenic development and delays germination of cultured immature embryos of barley.High level of sucrose (12-18%) also inhibits the germination of immature embryos.Other factors viz., reduced O2 tension, elevated temperature, ABA also inhibits the precocious germination

Precocious Germination contd….

Applications of embryo culture

In interspecific and intergeneric hybridization, incompatibility barrier often prevents the normal seed development and production of hybrids.To overcome the above barriers for obtaining the hybrids, the embryo culture technique is effective utilized in which the nutritional relationship between the embryo and endosperm is restored by providing the artificial medium to induce and complete growth of hybrids embryos and is called as embryo rescuing.Intergeneric hybrids have been obtained between Hordeum and Secale; Hordeum and Hordelymus, Triticum and Elymus; Triticum and Secale and Tripsacum and Zea.

1. Embryo rescue in wide crosses

Wide hybridization thru’ embryo rescueWide crosses Purpose

Corchorus capsularis x C. Olitorius

Hybrids had fibres with quality of C. capsularis and strength of C. olitorius

Hordeum vulgare x H. The hybrids possessed winter hardiness and mildew resistance like H. bulbosum

Lycopersicon esculentum x L. peruvianum

The hybrids possessed resistance to viruses, moulds and nematodes along with good fruit set like L. peruvianum

Melilotus officianalis x M. alba

Hybrids resembling M. officinalis in agronomic characters and low coumarin content like M. alba

Nicotiana tabacum x N. resophilia

To get plants with resistance to black shank

Oryza sativa x O. officinalis To transfer pest resistance

Trifolium pratense x T. sarosiense

To impart perennial plant habit to red clover

2. Shortening breeding cycle of plantsUseful in reducing the breeding cycle of new varieties in cases where long dormancy causes extension of breeding cycle. Cultivated varieties of rose generally take about a year to flower and two to three months for the formation of fruits. Seedlings produced from cultured embryos flower in two to three months. These flowers can serve as the male parent for further crosses enabling the breeder to produce two generations in one year or shortening the breeding cycle to three or four months. In weeping crap apple (Malus sps) the seeds cultured in vitro produce seedling in four months, on the other hand, seeds planted in the soil take about nine months to germinate.

In early ripening fruit cultivars the seeds do not germinate because their embryos are still immature.Using the embryo culture method it is possible to raise seedlings form the sterile seeds of stone fruits (sweet cherry), peach, apricot and plum.‘Makapuna” coconuts are realized for their soft fatty endosperm in place of liquid endosperm.Under normal conditions the seeds fail to germinate. But De Guzman et al. (1985) obtained 85% success in raising makapuna seedlings through embryo culture.Same techniques also used in Maranta, Colocassia, Musa bulbisima and Pinus armandii X P. koriensis

3. Overcoming seed sterility

4. Monoploid production

Production of haploids in barleyH. vulgar X H. bulbosum where fertilization occurs normally, but there after chromosome elimination of H. bulbosum resulting in the production haploid embryos carrying only barleychromosomes which could be rescued by embryo culture.In other crosses viz., Agropyron tsukuishiens X H. vulgare and other cereals haploids were produced following this technique.

5. MicropropagationBecause of their juvenile nature the embryos have high potential for regeneration.Complete plantlets in vitro from conifers were achieved in long leaved pines (Pinus palustris) through embryo culture.Both organogenesis and embryogenesis have been induced in major cereals and forage grasses from embryonic tissues.

6. Endosperm cultureIn 1947, LaRue, successfully cultured the corn endosperm and obtained plantlets with root-shoot axis and miniature leaves.The triploidy can be exploited in the crops viz. apple, banana, mulberry, sugar beet, tea and watermelon where seeds are not of commercial importance.

The embryo culture has been proved as a viable technique for resynthesising some of the plant hybrids For example Brassica napus has been resynthesised from the cross of B. campestris/B. oleracea using embryo culture. The recent approach is back crossing the resynthesised B. napus (2n=38) to B. campestris(2n=20), so that the genes from B. oleracea can be transferred to B.campestris . In 1988 Quazi made an attempt in this regard and came out with successful results. He got a line from the back crosses of (B. napus/B. oleracea)/B. oleracea which is resistant to cabbage aphid attack.

Embryo culture and back crossing in gene transfer

Other biotechnological usesThe embryo culture technique can be effectively engaged in seed testing of various tree species, germinating seeds of obligate phanerogamic parasites, Studying the host-pathogen relationship in seed-borne diseases and studying developmental embryogenesis. Genetic transformation using the desiccated zygotic embryos which were generally perforated with larger pores.Toper et al. (1989) transformed some cereals by imbibing mechanically isolated zygotic embryos in a solution containing plasmids carrying chimeric genes for successful transformation.This approach is a potential tool in transforming important horticultural crops in future.

Mature embryo culture in Cassava

Cassava fertility and seed viability are frequently low, which can be a disadvantage in a breeding programme. An embryo culture method in which embryonic axes are excised from mature seeds and placed on a culture medium containing 1.23 μl indolebutyric acid (IBA) at 30°C under continuous light. The number of plants recovered by embryo culture was much greater than the number recovered from conventional seed germination procedures

A rapid regeneration protocol for proembryos of Phaseolus angustissimus as young as 1 day after pollination (DAP) involving pod culture for 1 week followed by embryo culture for 2 weeks and embryo germination for 1 or 2 weeks is provided. Optimization of the media was conducted with pods collected 3 DAP.The best pod culture medium was composed of basal medium salts with vitamins, 1000 mg l-1 glutamine, 1000 mg l-1 casein hydrolysate 1.9 μM, 3% sucrose and 0.5% agar.Embryo culture medium consisted of basal medium with 500 mg l-1 glutamine, 250 mg l-1 casein hydrolysate, 1.9 μM ABA, 3% sucrose and 0.5% bacto-agar

Plant regeneration via pod culture, embryo culture and embryo germination for pods of P. angustissimus

For the first time Jie-ning Xiao et al. (2004), regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified MS medium with 5mg (25 mM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mg (23 mM) kinetin. Concluded that secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium.

Immature embryo culture in Mango

Immature embryo with seed

Proembryonic mass

Globular embryo

Mature somatic embryo

Plantlet dev. Form imm. embryo

An interspecific hybrid of the sexually incompatible species G. hirsutum cv.Laxmi and G. arboreum cv. Jyoti was obtained through in ovulo embryo culture. Eight to twelve-day-old ovules were excised and cultured on BS medium supplemented with IAA (5 ×10-6 to 7 × 10 -7M), Kinetin (5 X 10 -6 to 5 × 10- -8), GA (5 X 10 -7 to 5 × 10 -9), Ammonium chloride (5 to 15mM) and Casein hydrolysate (50 to 200mg/l) added individually and in various combinations along with sucrose. No single medium was adequate to ensure complete development of the fertilized ovules to plantlets, thus necessitating a sequential five step transfer to different media. Cytological studies confirmed the hybrid nature of the plants

Ovules of 85 D old

Enlarged ovule after 35 D of incubation

Triploid hybrid plant

2n = 39

Synthetic seed technology may be of value in breeding programs and allow the propagation of many elite genotype derived plants in a short time.A range of artificial endosperm treatments of Cleopatra tangerine zygotic embryos were evaluated for suitability for encapsulation of somatic embryos.An artificial endosperm was used to encapsulate somatic and zygotic embryos. After encapsulation, zygotic embryos germinated after four days of culture while somatic embryos germinated asynchronously after 20 days. Somatic embryo-derived plantlets showed greater vigour than zygotic embryo-derived plantlets. Results showed that this artificial endosperm is adequate for Cleopatra tangerine somatic embryo germination and conversion into plants

Encapsulation of embryos with Calcium arginate