Lect-8 In Vitro Pollination, Fertilization And Embryo Culture

In vitro Pollination & Fertilization ¾Reproductive isolation is considered to play a key part in evolution. ¾Plants and animals have developed a range of strategies that minimize gene flow between species. ¾In plants, these strategies involve either prezygotic barriers, such as differences in floral structure and pollen-stigma recognition (inhibition of pollen tube growth), or postzygotic 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 ¾ In most crop improvement programme often wide hybridization is resorted to transfer the genes for abiotic and biotic stress from alien genera. ¾ Involves interaverietal, inter-varietal, intergenera and inter-family crosses to transfer the target genes from the donors. ¾ Eg. Wheat & Rye (Inter-genera) Rice: Wide crosses of interaverietal Most failure in these crosses are due to i) self incompatibility, ii) cross incompatibility etc.

In vitro pollination & Fertilization ¾ 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 ¾ 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) ¾ (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. b. c. d. e.

Bud pollination Stump pollination Heat treatment of the style Irradiation and Mixed pollination

In vitro pollination ¾Development of seed through in vitro pollination of exposed ovules is termed as “Test tube fertilization” ¾Seed formation following stigmatic pollination is termed as “ in vitro pollination” ¾In 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. ¾IVF 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 (Tian and Russell, 1997).

In vitro pollination: Methodology Conditions required for successful IVF ¾ Ovaries should contain large number of ovules (In solanaceae many members viz., Nicotiana tabacum, N. alata, N. rustica and Petunia hybrida), and in Papavaraceae and cryophillaceae, the placenta are covered with several hundreds of ovules. ¾ Isolation of ovules with out any damage as possible in these spp. which contributing maximum to the success in the in vitro pollination. ¾ The other requirement is the pollen which should be viable and able to germinate. ¾ There must be abundant growth of pollen tubes all over the ovules and placenta in the culture.

Other requirements: Before to start, the information on 1. Time of anthesis 2. Time of dehiscence 3. Time of germination of pollen tubes into ovules 4. Viability of ovules and fertilization inside the embryo sacs etc. are essential for successful IVF.

Disinfection of materials ¾The buds to be brought to the laboratory for aseptic culture just before the anthers are at the stage of dehiscence ¾The 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) ¾The ovary wall should be carefully peeled with scalpel, needle to expose the mass of ovules attached to the placenta. ¾To perform stigmatic pollination the excised pistils are to be carefully surface sterilized without wetting the stigma.

Preparation of pollen & ovary for IVF ¾Anthers collected form the bud are kept in as sterile Petri dish containing a pre-sterilized filter paper until their dehiscence. ¾Generally the pollen deposited directly on the cultured part of the pistil performs better than that spread on the medium around the ovules. ¾In graminaceous family the ovaries are well protected by many layers of husks and hence the surface sterilization is not required. ¾In maize husks are severed after 2-4 days of silking with a scalpel. ¾Ovaries 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 ¾Pollination is done directly on the silks or the ovules in vitro. ¾Twenty four hours after the pollination silks are clipped off and the Petri plates sealed. ¾To 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 IVP ¾ The growth of the pollen tube on the barren ovule is affected by the presence of moisture on the surface of the ovule. ¾ The ovules may be wiped with a filter paper and then covered with pollen grains. ¾ After 4-6 days the ovules contain single celled zygote which requires a complex growth condition. ¾ 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. ¾ Afterwards they can be transferred to a fresh medium without placenta ¾ Ovule culture is proved useful in raising interspecific hybrids in Gossypium, Helianthus and Trifolium

Ovary Culture after Pollination ¾ Nitsch (1951) developed the in vitro technique for the ovary culture and successfully cultured the ovaries of Cucumis and Lycopersicum. ¾ Addition of vitamin B to the medium resulted in the development of normal fruits and viable seeds ¾ Enrichment of medium with IAA and coconut milk induced larger fruits than the fruits formed in in vivo condition (Kanta & Maheswari, 1963). ¾ The floral envelops (lemma & palea) play an important role in the development of fruit & embryo in monocots. ¾ Ovaries excised soon after the fertilization in Triticum aestivum & T. spelta develop in the culture only when the floret envelops remain intact. ¾ 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… ¾ In the absence of the hull factor, DNA synthesis and cell elongation in barley embryo cells takes place but cell division does not occur. ¾ Similarly the association of perianth in dicots has been found necessary for the development of fruits. ¾ 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 ¾1. Physiological status of the explant ¾2. Culture medium ¾3. Storage condition ¾4. Genotype

Physiological status of the explant ¾ The physiological state of the pistil at the time of excising the ovules or ovary highly influence the seed set after the IVP. ¾ 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…. ¾Pollen germination & growth of pollen tube through the style may influence the synthesis of some protein which may prevent the entry of pollen tube into the ovule. ¾Hence it is necessary to find which part of the pistil, the barrier exists. ¾To 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 condition ¾The time of excising the ovules from pistil has a definite influence on seed set after IVP. ¾Ovules excised 1-2 DAA showed higher seed set than on the day of anthesis. ¾In maize spikes pollinated 3-4 days after silking yields better results.

2. Culture medium ¾ Nitsch’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). ¾ Addition of kinetin and CH (Casein hydrolysate) was essential to promote the initial growth of the embryo. ¾ Several orchid ovules grew successfully in a simple 10% sucrose solution. ¾ But the ovules of Zephyranthes required coconut milk or casamino acid in the Nitsch’s medium.

2. Culture medium contd…… ¾ In Trifolium ripens, ovules (1-2 DAP) required the supplementation of the medium with juice prepared from young fruits of cucumber or water melon. ¾ GA3 (10 Mg l-1) in addition to fruit juice further improved the seed development in the young ovules ¾ Steward & Hsu, (1978) developed a medium for raising intraspecific & inter-specific hybrids from young ovules ¾ Presence of 10 μg l-1 IAA or kinetin improve the number of seed per ovary. ¾ 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. ¾ Osmolarity of the medium also affects the development of excised ovules. ¾ Normal sucrose 4-10%. But a ovule with zygote with few endosperm nuclei- 6%; ovules just after fertilization - 8% sucrose.

3. Storage conditions ¾ No precise information on the effect of temperature and light on the test tube fertilization ¾ Usually the first step of the process occur at room temperature without special lighting. ¾ Later the ovaries are transferred to 22-26°C

4. Genotype ¾Pollen grains of crucifer are difficult to germinate and a modified technique is required ¾Dipping Brassica ovules in 1% CaCl2 before pollination followed by transfer to Nitsch’s medium is required to successful seed set.

Application of in vitro pollination Four different major areas are: 1. Overcoming self-incompatibility 2. Overcoming cross-incompatibility 3. Haploid production through parthenogenesis 4. 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 ¾ Petunia axillaris and P. hybrida are self incompatible species. ¾ Barrier to the germination of pollen exists in the ovary. ¾ This was overcome by IVP and seed set has been reported. ¾ In Petunia the self incompatibility can also be overcome by bud pollination ¾ Several interspecific, intergeneric and interfamilial crosses were attempted through in vitro ovular, placental pollination.

++ Abundant germination 3: embryo formed

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

Germinating pollen tubes

Twin Embryo 3 d after in vitro pollination

Interspecific, intergeneric and interfamilial crosses attempted through IVP

3. Haploid production through in vitro pollination ¾ Haploid production through in vitro pollination in Mumulus luticus when pollinating its exposed ovules with Torena founieri was reported. ¾ The haploid of M. luticus developed parthenogenetically which otherwise could not have been obtained through anther culture. ¾ Such parthenogenetic development of haploids also been reported in Hordeum vulgare, Nicotiana tabacum & Triticum aestivum

¾Haploid plantlet regeneration through gynogenesis in Citrus clementina cv. Nules, induced by in vitro pollination with pollen grains of Oroblanco, a triploid cultivar of grapefruit. ¾It indicates that parthenogenesis induced in vitro by triploid pollen can be an alternative method to obtain haploids in monoembryonic cultivars of Citrus. ¾Pollination and mature stage of pistils was necessary for gynogenic embryo regeneration. ¾Fourteen haploid gynogenic embryos of Nules clementine were obtained

Stigma exudate and Oroblanco pollen grains

1 month old pollinated pistil

Germination of a haploid embryo

Germination of a haploid embryo

Gynogenic embryos breaking through the ovary

4 months old pollinated pistil

A haploid citrus plant obtained through in vitro pollination

Haploid production through IVP in wheat ¾ To improve haploid plant production in durum wheat the haplo-method involving intergeneric crossing with maize followed by embryo rescue was used. ¾ The wheat genotype was significant for ovary development, embryo and plant formation, whereas the maize genotype was significant only for embryo formation ¾ The significant effect of the wheat genotype on embryo formation was found. ¾ 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

¾ 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. ¾ 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 ¾ The embryos obtained from wheat X I. cylindrica crosses 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-cysteine and L-leusine, 30 g/l sucrose and 8 g/l agar agar. ¾ 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 ¾ Maize plants tolerant to heat can be produced through in vitro pollination at high temperature. ¾ Pollen grain at temperature 38ºC were able to effect fertilization and the resulting maize plants expressed the trait for heat tolerance. ¾ Additionally these plants exhibited increased vigour and grain yield. ¾ 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 IVF ¾Difficulties in isolating gametes of higher plants have impeded our understanding of gamete physiology, activation of development and early embryogenesis in flowering plants. ¾However increasing number of tools now available to manipulate male and female gametes of higher plants provides numerous opportunities for scientific and biotechnological progress (Fig.). ¾Isolated 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 Technique ¾ In angiosperms, the embryo is the miniature sporophyte resulting from the fertilized egg or zygote and the endosperm is the main nutritive tissue for the embryo. ¾ 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. ¾ The culture of embryo and endosperm has been practiced by plant breeders for over half a century. ¾ Among the two techniques, the former has a long list of success stories whereas the latter has very narrow success

Embryo culture ¾In seed bearing plants, embryos are easily accessible. ¾They can be separated with relative ease from the maternal tissues and cultured in vitro under aseptic conditions in media of known chemical composition. ¾Hanning (1904) first reported his systematic attempt to culture isolated embryo of Cochleria and Raphanus (Cruciferae). ¾He successfully raised seedling from the cultured embryo using semi-solid medium containing mineral salts & sugar. ¾Later 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 culture ¾ According to Pierik (1989) there are two types of embryo culture ¾ 1. Culture of immature embryo: ¾ Mainly used to grow immature embryos of hybrids which fails to germinate which require a complex medium. ¾ Success of this culture mainly depends upon the developmental stage of the immature embryo ¾ By 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 embryo ¾ Mature embryos are excised from the seeds and cultured mainly to avoid inhibition in the seed germination. ¾ This type of culture is relatively easier as embryo require a simple nutrient medium containing mineral salts, sugar and agar.

Embryo culture techniques ¾ 1. Disinfection ¾ Embryos develop normally inside the ovules which in turn covered by ovaries ¾ Since they are already in a sterile environment they do not require disinfection unless they are injured or seriously infected. ¾ 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. ¾ 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 material ¾ By immersing the material in hypochlorite containing commercial bleach (5-10% Chlorox, 0.45% sodium or calcium hypochlorite) for 5-10 min. ¾ Or ethanol (70%) for 5 min. ¾ 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. ¾ 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 embryos ¾ Excision operation should be performed under aseptically in a laminar air flow hood. ¾ A stereomicroscope (90X) with a cool ray fluorescent lamp is required for excising smaller embryos. ¾ Common tools used are: Forceps, dissecting needles, scalpels, razor blades and Pasteur pipettes ¾ Mature embryos can be excised easily by splitting open the seed. ¾ 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 transplant ¾ Abortion of embryos at early stage of development is due to the non availability of nutrients. ¾ 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. ¾ 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. ¾ Generally endosperm older than the embryo by 5 days was more efficient as a nurse tissue for the growing 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

Hybrid embryo

Normal endosperm

Culture Medium-Nutrition requirement ¾ The requirement of culture medium depends on the types of embryo culture. ¾ They may be either post-germinal or pre-germinal. ¾ In the case of post-germinal embryo culture, embryos are cultured only to speed up the process after germination. ¾ This can be achieved with less complex medium or even with sucrose or glucose solution. ¾ In pre-germinal embryo culture, immature embryos are cultured to get plantlets, where the embryos require a complex nutrient medium. ¾ Includes modifications in the composition of mineral salts, organic nutrients and growth regulators

Nutrition requirement contd….. ¾ The composition of the culture medium has to be formulated to suit the developmental phase of the embryo. ¾ 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 ¾ Embryos excised at near maturity stage are completely autotrophic and grow on simple medium comprising salts with a carbohydrate source. ¾ Immature embryo which are heterotrophic requires complex medium including vitamins, plant extracts and plant growth regulators.

¾ Monnier (1976) developed technique which allows complete development of Capsella embryo (early globular stage) upto germination without moving them from the original position in the culture plate as below:

Mineral salts ¾ Inorganic nutrients of MS, B5 and White’s media with certain degree of modification are the most commonly used media for embryo culture. ¾ Monnier (1976) modified the MS medium composition for higher survival rate of cultured immature embryo. ¾ 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…

Carbohydrates ¾ Sucrose is the most commonly used source of energy for embryo culture ¾ In maize, addition of maltose, lactose, raffinose, or mannitol may be required. ¾ Superiority of Glucose over sucrose is reported in the embryo growth of Carex lucida, several spp of Rosaceae and Lilium hybrids. ¾ Glucose & sucrose besides nutrition, also maintain osmolarity of the culture medium. ¾ Mature embryo grow fairly well at low sucrose, but younger embryos require higher conc. of sucrose. ¾ Various concentrations of sucrose used for different species band age of embryo is given below:

Role of sucrose in the medium

Nitrogen & Vitamins ¾ Embryos have enzyme system to reduce nitrates to ammonium ¾ Ammonium nitrate is superior to sodium nitrate, potassium nitrate and ammonium diphosphate ¾ Presence of ammonium is essential for the growth & differentiation of embryos ¾ Hanning (1904) reported that asparagine enhanced embryo growth ¾ Glutamine to be a superior source of N in Capsell sp. ¾ CH (a amino acid complex) widely used as an additive to embryo culture media. ¾ Optimum level of CH is 500 mg l-1.

Natural Plant Extracts ¾ Coconut 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. ¾ CM stimulates the growth of young excised embryos of sugar cane, barley, tomato, carrot, interspecific hybrids of Vigna and fern spp. ¾ Water extracts of dates, bananas, hydrolysates of wheat gluten and tomato juice were reported to promote growth of embryos. ¾ Alcohol diffusates of young seeds of Datura and Sechium had comparative effect of CM and Lupinus has got twice the effect of CM.

Growth Regulators ¾ Auxins & cytokinins are not generally used since they induce callus formation. ¾ At very low level (0.01 mg l-1), GA promotes embryogenesis of young barley embryos (without inducing precocious germination) ¾ ABA (abscisic acid) also has similar effects on barley and Phaseolus embryos

pH of the medium ¾ Embryos grow well in a medium with pH of 5-7.5 ¾ Generally the medium pH is adjusted 0.5 above the desired pH to compensate the uncontrollable change during autoclaving process

Incubation conditions ¾ Incubation temperature of 25 ±2°C normally supports the growth of majority of embryos. ¾ Sometimes the incubation temperature may vary with the genotypes of the same species ¾ Zennia, Phaseolus and cotton adopted to warm temperature (27- 30°C) to culture embryos. ¾ Whereas in Brassica hybrids, rice, barley in cold regions or seasons require temperature of 17-22°C (Hu & Wang, 1986).

Light ¾It 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. ¾A 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. ¾This suggests that initial incubation of embryos for 4 days is essential.

Role of suspensor in embryo culture ¾ The 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. ¾ Generally, it is difficult to excise the suspensor along with the embryo because of its small size and delicate structure. ¾ Older embryos (500 μm or more in length) appear to grow well with or without a suspensor ¾ Observations 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. ¾ The requirement of the suspensor may be substituted by the addition of GA or ABA to the culture medium

Precocious Germination ¾ Plant physiologists and biochemists conceive embryo development as a linear progression from zygote formation to germination. ¾ According to Walbot (1978), embryo development can be classified into five stages (Table 11.6).

Precocious Germination contd…. ¾ Excised embryos on a nutrient medium tend to bypass the stage of dormancy. ¾ Directly develop into a weak seedling. ¾ This phenomenon of seedling formation without completing normal embryogenic development is called precocious germination. ¾ CH promotes further embryogenic development and delays germination of cultured immature embryos of barley. ¾ High level of sucrose (12-18%) also inhibits the germination of immature embryos. ¾ Other factors viz., reduced O2 tension, elevated temperature, ABA also inhibits the precocious germination

Applications of embryo culture 1. Embryo rescue in wide crosses ¾In interspecific and intergeneric hybridization, incompatibility barrier often prevents the normal seed development and production of hybrids. ¾To 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. ¾Intergeneric hybrids have been obtained between Hordeum and Secale; Hordeum and Hordelymus, Triticum and Elymus; Triticum and Secale and Tripsacum and Zea.

Wide hybridization thru’ embryo rescue Wide 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 plants ¾Useful in reducing the breeding cycle of new varieties in cases where long dormancy causes extension of breeding cycle. ¾Cultivated varieties of rose generally take about a year to flower and two to three months for the formation of fruits. ¾Seedlings produced from cultured embryos flower in two to three months. ¾These 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. ¾In 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.

3. Overcoming seed sterility ¾In early ripening fruit cultivars the seeds do not germinate because their embryos are still immature. ¾Using the embryo culture method it is possible to raise seedlings form the sterile seeds of stone fruits (sweet cherry), peach, apricot and plum. ¾‘Makapuna” coconuts are realized for their soft fatty endosperm in place of liquid endosperm. ¾Under normal conditions the seeds fail to germinate. ¾But De Guzman et al. (1985) obtained 85% success in raising makapuna seedlings through embryo culture. ¾Same techniques also used in Maranta, Colocassia, Musa bulbisima and Pinus armandii X P. koriensis

4. Monoploid production Production of haploids in barley ¾H. vulgar X H. bulbosum where fertilization occurs normally, but there after chromosome elimination of H. bulbosum resulting in the production haploid embryos carrying only barley chromosomes which could be rescued by embryo culture. ¾In other crosses viz., Agropyron tsukuishiens X H. vulgare and other cereals haploids were produced following this technique.

5. Micropropagation ¾Because of their juvenile nature the embryos have high potential for regeneration. ¾Complete plantlets in vitro from conifers were achieved in long leaved pines (Pinus palustris) through embryo culture. ¾Both organogenesis and embryogenesis have been induced in major cereals and forage grasses from embryonic tissues.

6. Endosperm culture ¾In 1947, LaRue, successfully cultured the corn endosperm and obtained plantlets with root-shoot axis and miniature leaves. ¾The triploidy can be exploited in the crops viz. apple, banana, mulberry, sugar beet, tea and watermelon where seeds are not of commercial importance.

Embryo culture and back crossing in gene transfer ¾The embryo culture has been proved as a viable technique for resynthesising some of the plant hybrids ¾For example Brassica napus has been resynthesised from the cross of B. campestris/B. oleracea using embryo culture. ¾The 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 . ¾In 1988 Quazi made an attempt in this regard and came out with successful results. ¾He got a line from the back crosses of (B. napus/B. oleracea)/B. oleracea which is resistant to cabbage aphid attack.

Other biotechnological uses ¾The embryo culture technique can be effectively engaged in seed testing of various tree species, germinating seeds of obligate phanerogamic parasites, ¾Studying the host-pathogen relationship in seedborne diseases and studying developmental embryogenesis. ¾Genetic transformation using the desiccated zygotic embryos which were generally perforated with larger pores. ¾Toper et al. (1989) transformed some cereals by imbibing mechanically isolated zygotic embryos in a solution containing plasmids carrying chimeric genes for successful transformation. ¾This approach is a potential tool in transforming important horticultural crops in future.

Mature embryo culture in Cassava ¾ Cassava fertility and seed viability are frequently low, which can be a disadvantage in a breeding programme. ¾ 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. ¾ The number of plants recovered by embryo culture was much greater than the number recovered from conventional seed germination procedures

¾ 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. ¾ Optimization of the media was conducted with pods collected 3 DAP. ¾ 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. ¾ 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% bactoagar

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

Immature embryo culture in Mango ¾ 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. ¾ 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). ¾ Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mg (23 mM) kinetin. ¾ Concluded that secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium.

Plantlet dev. Form imm. embryo

Immature embryo with seed

Proembryonic mass

Globular embryo

Mature somatic embryo

¾ An interspecific hybrid of the sexually incompatible species G. hirsutum cv.Laxmi and G. arboreum cv. Jyoti was obtained through in ovulo embryo culture. ¾ 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. ¾ 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. ¾ 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

¾Synthetic seed technology may be of value in breeding programs and allow the propagation of many elite genotype derived plants in a short time. ¾A range of artificial endosperm treatments of Cleopatra tangerine zygotic embryos were evaluated for suitability for encapsulation of somatic embryos. ¾An artificial endosperm was used to encapsulate somatic and zygotic embryos. ¾After encapsulation, zygotic embryos germinated after four days of culture while somatic embryos germinated asynchronously after 20 days. ¾Somatic embryo-derived plantlets showed greater vigour than zygotic embryo-derived plantlets. ¾Results showed that this artificial endosperm is adequate for Cleopatra tangerine somatic embryo germination and conversion into plants

Encapsulation of embryos with Calcium arginate