INTRODUCTION Successful sexual hybridization involves a series of events including pollen germination, pollen-tube growth, fertilization, embryo and endosperm development and seed maturity, leading to germination (Debbarama et al, 2013). These events can be hampered by somatoplastic sterility, cytoplasmic-genic male sterility and structural differences in chromosomes. A very important and useful biotechnological tool for raising hybrid progeny in intractable crosses is embryo rescue. A given cross may fail for any of a number of reasons: seedlessness in either or both parents, inimical influence of endosperm resulting in inhibited embryo development, poor fruit-set / heavy fruit-drop at initial stages of the cross, distant crossing (interspecific / intergeneric / intervarietal breeding resulting in embryo abortion), genetic incongruity of parental genomes, mismatch in ploidy levels, etc. Embryo culture is one of the earliest forms of in vitro culture applied to practical problems and is probably the tissue culture technique that has proven of greatest value to breeders (Dunwell, 1986). At the Indian Institute of Horticultural Research, Bangalore, the authors have FOCUS J. Hortl. Sci. Vol. 8(1):1-20, 2013 Hybrid embryo rescue: a non-conventional breeding strategy in horticultural crops Leela Sahijram, Jaya R. Soneji1, Anitha Naren2 and B. Madhusudhana Rao Division of Biotechnology Indian Institute of Horticultural Research Hessaraghatta, Bangalore 560 089, India E-mail: leelas@iihr.ernet.in ABSTRACT Production of interspecific and intergeneric hybrids is useful for transfer of desirable genes from wild species into cultivated species. In many instances, progeny from wide crosses is difficult to produce owing to several barriers. Post-zygotic barriers such as endosperm abortion and, at later stages, embryo degeneration are of common occurrence, leading to low fertility; but these have been overcome through the use of embryo rescue and several hybrids have been developed. This approach is especially useful in horticultural crops, more so in fruit crops. In our laboratory, we have developed protocols for hybrid embryo rescue in several top-of-the-line fruit crops that suffer from an inability to cross naturally (e.g., distant crosses, use of seedless parent/s) or instances where initial fruit drop is very high. Thus, interspecific, intergeneric and intervarietal hybrids have been generated in mango, banana, seedless grape, papaya and seedless citrus using embryo rescue. Culture of embryos has also been demonstrated in rose, capsicum, hot pepper, onion and tomato. Among the very important strategies under non-GM biotechnologies figure techniques of hybrid embryo rescue, and related applications like ovule / ovary / placental cultures through sequential embryo culture. Embryo culture applied to practical problems is a tissue culture technique that has proven to be of greatest value to breeders. Key words: Hybrid embryo rescue, horticultural crops, in vitro culture, non-conventional breeding successfully developed technologies using excised hybrid embryo / sequential embryo culture in a number of horticultural crops (Leela Sahijram, 2007, 2009, 2011). The authors have raised hybrids in seedless grape & lime, in difficult crosses of papaya, and in mango, banana, rose, etc. The underlying principle of embryo rescue technique is aseptic isolation of the embryo and its transfer to a suitable medium for facilitating its development under optimal conditions. The potential these techniques hold is a viable alternative to parasexual hybridization and somatic embryogenesis (Stewart, 1981). Failure to produce a hybrid may be due to either pre- or post-fertilization incompatibility. If fertilization is possible between two species or genera, the hybrid embryo may abort before maturation. If fertilization does occur, the embryo resulting from an interspecific or intergeneric cross can oftentimes be rescued and cultured to produce a whole plant. Such a method is referred to as embryo rescue. It is a tissue culture technique used in various breeding programs engaged in creating new genotypes. The procedure involves dissection of the developing embryo out from its environment Present address: 1Polk State College, Department of Biological Sciences, Winter Haven, Florida 33881, USA 2Shri Murugappa Chettiar Research Centre, Taramani, Chennai 600 113, India 2 and culturing it on a synthetic medium. In certain instances, ovule culture may need to be applied preceding excised embryo culture. Plant breeders usually rescue inherently weak, immature or hybrid embryos to prevent their degeneration. Successful production of plants from cultured embryos depends largely upon maturation stage and composition of the medium besides, of course, the genotype. Abortion of embryos at one or the other stage of development is a characteristic feature of distant hybridization. Hybrid embryo rescue is a popular approach for raising hybrids. Currently, embryo rescue holds great promise for not only effecting wide crosses but also for obtaining haploid plants, and for shortening breeding cycle in presence or absence of a protracted dormancy. THE TECHNIQUE OF EMBRYO CULTURE Embryo culture is the sterile isolation and growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant. The first attempt to grow embryos of angiosperms was made by Hannig in 1904 (Bridgen, 1994) who obtained viable plants from in vitro isolated embryos of two crucifers Cochleria and Raphanus. In 1924, Dietrich grew embryos of different plant species and established that mature embryos grew normally but those excised from immature seeds failed to achieve the organization of a mature embryo. They grew into seedlings, skipping the stages of normal embryogenesis and without completion of the dormancy period. Laibach (1925, 1929) demonstrated practical application of this technique by isolating and growing the interspecific cross Linum perenne x L. austriacum that otherwise aborted in vivo. Embryo culture is now a well-established and useful branch of plant tissue culture and the subject has been reviewed by researchers (Sharma et al, 1996; Reed, 2004; Raghavan, 2003; Taji et al, 2002; Leela Sahijram, 2007, 2010; Bhojwani et al, 1999; Bhojwani and Razdan, 1996; Chadha et al, 2000; Chawla, 2002). There are broadly two types of embryo culture: (i) Mature embryo culture is the culture of mature embryos derived from ripe seeds. This type of culture is done when embryos do not survive in vivo or become dormant for long periods of time, or, is done to eliminate inhibition of seed germination. Seed dormancy of many species is due to chemical inhibitors or mechanical resistance present in structures covering the embryo (rather than dormancy of the embryonic tissue). Excision of embryos from the testa and culturing them in nutrient media may bypass such seed dormancy. Some species produce sterile seeds (which may be due to incomplete embryo development). Embryo culture procedures may yield viable seedlings. Embryos excised from the developing seed at or near the mature stage are autotrophic and can be grown on a simple inorganic medium with a supplemental energy source. Seeds having hard seed coats are sterilized and soaked in water for a few hours to a few days. Sterile seeds are then split open and the embryos excised out. (ii) Immature embryo culture, also known as embryo rescue, is the culture of immature embryos to rescue embryos from wide crosses, crosses involving seedless parent/s, or where fruit fall is heavy in early stages of embryo development. This is mainly used to avoid embryo abortion, with the purpose of producing a viable (hybrid) plant. Wide hybridization, where individuals from two different species of the same genus or different genera are crossed, often leads to failure of the cross. There are several barriers which operate at pre- and post-fertilization levels to prevent successful gene transfer from wild into cultivated species. Pre-fertilization barriers include all factors that hinder effective fertilization, which is usually due to inhibition of pollen-tube growth by the stigma or upper style. Post- fertilization barriers hinder or retard development of the zygote after fertilization and inhibit normal development of the seed. This frequently results from failure of the hybrid endosperm to develop properly, leading to starvation of the hybrid embryo or results from embryo-endosperm incompatibility where the endosperm produces toxins that kill the embryo. Raghavan (1976) discussed evidence suggesting that embryos of inviable hybrids possessed a potential for initiating development, but were inhibited from reaching adult size with normal differentiation. Endosperm failure generally results in eventual starvation of the abnormal embryo. Isolation and culture of hybrid embryos prior to abortion circumvents these strong post-zygotic barriers. Production of interspecific and intergeneric hybrids is the most impressive and conspicuous application of embryo rescue and culture technique, particularly, for subsequent valuable gene transfer from wild species. There are normally no problems of disinfection of embryos in such cultures. Florets are removed at the proper time, and, either the florets or the ovaries are sterilized. Ovules can then be removed from the ovaries. The tissue within the ovule in which the ovule is embedded is already sterile. For mature-embryo culture, J. Hortl. Sci. Vol. 8(1):1-20, 2013 Leela Sahijram et al 3 either single, mature seeds are disinfected or, if the seeds are still unripe, then the still-closed fruit is disinfected. The embryos can then be aseptically removed from the ovules. The most important aspect of embryo culture work is selection of the medium necessary to sustain continued growth of the embryo. Younger the embryo, more stringent is its requirement. Although mature embryos can be grown on basal salt-media with a carbon-energy source such as sucrose, young embryos, in addition, require various vitamins, amino acids and growth regulators and, in some cases, natural endosperm extracts. Young embryos should be transferred to a medium with high sucrose concentration (8-12%) which approximates the high osmotic potential of intracellular environment of the young embryo-sac, and, a combination of hormones that supports growth of heart-stage embryo (i.e., a moderate level of auxin and a low level of cytokinin). Reduced organic nitrogen in the form of aspargine, glutamine or casein hydrolysate is always beneficial. Malic acid is often added to the culture medium. After 1-2 weeks (when the embryo ceases to grow), it must be transferred to a second medium with normal sucrose concentration, low levels of auxin and a moderate level of cytokinin, which allows for renewed embryo growth, with direct shoot growth in many cases. Where the embryo does not show direct shoot formation, it can be transferred to a medium for callus induction, followed by shoot induction. When the embryos have grown into plantlets in vitro, they are generally transferred to sterile soil and grown to maturity under greenhouse conditions. Applications of embryo culture: Prevention of abortion in wide crosses: Development of intergeneric hybrids is possible: Raphanus sativus x Brassica napus; Actinidia deliciosa x A. eriantha; Actinidia deliciosa x A. arguata. Carica and Citrus also produced distant hybrids from their relatives using embryo rescue. In peach, a cross between an early Japanese cultivar Sunago Wase and a Chinese cultivar Yuhualu resulted in ‘Zaoxialu’ – an extra-early maturing hybrid (55 days to maturity, in Hanzhou, China). Some hybrids obtained through embryo rescue have recombined desirable genes like disease- and pest-resistance in various agricultural crop species (Table 1), besides inducing earliness. Embryo rescue is also used to recover crosses between diploids and tetraploids. Production of haploids: Embryo culture can be used for production of haploids or monoploids. Kasha & Rao (1970) developed a technique to produce barley monoploids. Interspecific crosses are made with Hordeum bulbosum as the pollen parent, and the resulting hybrid embryos are cultured. But, these exhibit H. bulbosum chromosome- elimination, resulting in monoploids of the female parent H. vulgare. Overcoming or breaking seed-dormancy: Seed dormancy can be caused by numerous factors, including endogenous inhibitors, specific light-requirements, low temperature, dry-storage requirements and embryo immaturity. Seeds of many species exhibit orthodox dormancy which, is, inhibition of germination by presence of certain plant hormones in tissues surrounding the embryo within a seed. Examples of such species include apples, pears, peaches, cedars, hemlocks, pines, firs, maples and roses. Dormancy can be a vexatious problem for growers and hybridizers alike. Embryo culture allows rapid testing of seed viability where seed dormancy can be circumvented. Removal of pericarp and testa from around the embryo removes the source of hormones that inhibit germination and, if all else is in place, allows the embryo to germinate and grow. It is a well-established technique, but, many previously-published protocols for embryo culture have complex and expensive culture requirements. Seed dormancy in Iris is due to the presence of a stable chemical inhibitor in the endosperm. In American basswood (Tilia americana), the seed is borne within a tough, indehiscent pericarp where the resistance is mechanical. Naturally-vegetatively propagated plants like bananas and Colocasia produce seeds that do not germinate Table 1: Resistance traits transferred to a variety of agricultural crops using embryo rescue S.No. Species crossed Resistance trait/s 1 Lycopersicon esculentum x Virus, fungi, nematodes L. peruvianum 2 Solanum melongena x Brinjal fruit & shoot borer S. khasianum (Leucinodes arbonlis) 3 Solanum tuberosum x Potato leaf-roll virus S. etuberosum 4 Brassica oleracea x Triazene resistance B. napus 5 Brassica napus x Cabbage aphid B. oleracea (kale) 6 Triticum aestivum x Salt-tolerance Thinopyrum scirpeum 7 Hordeum sativum x Powdery mildew and Spot-blotch H. vulgare 8 Hordeum vulgare x Powdery mildew H. bulbosum 9 Oryza sativa x O. minuta Blast (Pyricularia grisea); Bacterial blight (Xanthomonas oryzae) J. Hortl. Sci. Vol. 8(1):1-20, 2013 Hybrid embryo rescue in horticultural crops 4 in nature, probably due to recalcitrant dormancy. These stand to benefit from embryo culture (Leela Sahijram and Doreswamy, 2000). Shortening the breeding cycle: Species that exhibit seed dormancy factors localized in the seed-coat, pericarp, endosperm, or all three benefit from removal of these inhibitors to promote seed germination, eg., Brussels sprout, rose, apple, oil palm and Iris. Hollies (Ilex) embryos remain in the immature heart stage even though the fruits may have reached maturity. It takes three years for Ilex opaca seeds from mature berries to complete their embryonic development and to begin germination. Through embryo rescue, hybrid plants from this can be obtained in 2-3 weeks. Rosa normally takes a year to come to flowering; through embryo culture it was shown that it was possible to produce two generations in a year. Prevention of embryo abortion with early-ripening stone fruits: Early-ripening varieties of peach, cherry, apricot and plum produce sterile seeds (Ramming, 1985) which do not germinate under natural conditions and, eventually, decay in the soil. Seed-sterility here may be due to incomplete embryo-development. In early-ripening stone fruits, transport of water and nutrients to the immature embryo is cut off too soon sometimes, resulting in embryo abortion. Precocious germination has been prevented in Prunus through ovule culture, where the integument acts as a natural inhibitor (Ramming, 1985). ‘Makapuno’ coconuts are prized for their characteristic soft endosperm which fills the whole nut. These nuts fail completely to germinate because the endosperm invariably rots before the germinating embryo can emerge out of the shell. Embryo culture has been practiced as a general method in horticultural crops including avocado, peach, nectarine and plum. Two cultivars, ‘Goldcrest peach’ and ‘Mayfire nectarine’ have resulted from embryo culture and are now commercially grown. Embryos for propagation/ conservation/ germplasm exchange: Embryos are excellent material for in vitro preservation (Leela Sahijram and Rajasekharan, 1998) and propagation (Leela Sahijram and Doreswamy, 1989; 2000; 2004a), especially in conifers and in Gramineae family. As per IPGRI, Rome, it is compulsory to exchange germplasm of coconut internationally using embryo cultures (otherwise, shipping of whole nuts is cumbersome besides requiring huge cargo-space on flights or ships). Germination of seeds of obligatory parasites: Parasitic plants produce seeds that can be germinated to produce plantlets only on their chosen host-plant. Germination of seeds of obligate parasites in vivo without the host plant is impossible, but is achievable with embryo culture. Insectivorous plants have also been shown to propagate successfully on a synthetic medium using these techniques. EMBRYO RESCUE IN FRUIT CROPS Embryo rescue techniques in fruit crops have played an important role in breeding new, early-maturing seedless triploid types as well as obtaining distant hybrids preventing embryo degeneration at early development stage/s and shortening breeding cycle. Application and progress of embryo rescue in fruit crop breeding was reviewed and several problems, e.g., suitable culture-period, nutrient and environment conditions studied (Yi Hualin et al, 2001). With interspecific crosses, intergeneric crosses, and crosses between diploids and tetraploids, the endosperm often develops poorly or not at all. A synthetic medium can compensate for lack of support from endosperm. Mango Mango (Mangifera indica L.) is the leading fruit crop of India. In controlled crosses in breeding programmes of this crop, fruit-set in relation to the original number of flowers pollinated is extremely low, sometimes as low as 0.01% (Iyer, 1991). Therefore, our aim was to develop an embryo rescue based technology for improving breeding success. The mango has long been known to be a difficult system in traditional breeding programmes (Brooks, 1912; Mukherjee et al, 1968; Singh et al, 1980) because of certain of its inherent characteristics which include: 1. Long juvenile phase 2. High level of heterozygosity resulting in unpredictable outcome in hybridization 3. Heavy fruit-drop leading to low retention of crossed fruits 4. One seed per fruit 5. Polyembryony in some cultivars 6. Large acreage required for meaningful assessment of hybrids Classically, barring a few hybrid varieties resulting from planned hybridization programmes, almost all known cultivars have resulted from selection of chance seedlings from natural cross pollination. In Florida and South Africa, for example, none of the cultivars developed has come from a breeding programme. J. Hortl. Sci. Vol. 8(1):1-20, 2013 Leela Sahijram et al 5 Modern horticultural and industrial requirements in mango breeding (Iyer and Degani, 1997) emphasize: 1. Precocious bearing 2. Dwarf tree habit 3. Heavy and regular bearing 4. Freedom from physiological disorders 5. Resistance to major pests and diseases 6. Good shipping qualities and shelf-life 7. Ideal tree architecture Floral biology and pollination in mango The total number of flowers in a panicle may vary from 1000 to 6000 (Mukherjee, 1953). Initial fruit-set in mango is directly related to the proportion of perfect (hermaphrodite) flowers to staminate flowers, although, the final fruit-set does not necessarily depend on this ratio. This proportion becomes critical for optimum fruit-set in a cultivar when the proportion drops to 1%. In self-pollinated cultivars like ‘Dashehari’, ‘Langra’, ‘Chausa’ and ‘Bombay Green’, embryological studies have shown that although fertilization takes place after self- pollination, degeneration of endosperm occurs 15 days post- pollination in the self-incompatible parent. The self- incompatibility system operating in mango therefore appears to be of the sporophytic type. Leela Sahijram and Doreswamy (1987, 1988) initiated work on hybrid embryo culture and nucellar embryogeny in mango. In vitro hybrid embryo rescue was demonstrated successfully in mango (Mangifera indica L.) breeding by Leela Sahijram and Doreswamy (1990, 1991, 1992) and Leela Sahijram et al (2005). Immature hybrid embryos from controlled crosses involving cvs. Amrapali, Alphonso and Arka Anmol as the female parent and ‘Kerala Dwarf’ as the male parent were successfully rescued using excised embryo culture. Leela Sahijram et al (2000) showed that determination of hybrid embryo success rate in mango was genotype-dependent while Leela Sahijram and Ravindra (1997) worked out protocols for maturation of immature zygotic embryos from an intervarietal cross involving cvs. Amrapali and Kerala Dwarf. Breeding objectives in mango: Developing cultivars with (i) Regular bearing (ii) Dwarf tree habit with precocity in bearing (iii) Attractive, good-sized (300-500g), shape, good quality fruits (taste, flavour and firm flesh without fibre), high pulp:stone ratio With regard to improvement of rootstocks by breeding, the main desirable features are: 1. Polyembryony (a recessive trait) 2. Dwarfing influence on the scion 3. Tolerance to adverse soil conditions (pH, calcareous soil, etc.) 4. Good scion-compatibility In the late 1980s, the authors initiated embryo rescue work the on controlled cross ‘Mulgoa’ X ‘Neelum’ and successfully obtained hybrid plantlets in vitro. However, the hybrids on transfer to soil could not survive for more than four months. With this experience, another strategy was applied to the further crosses carried out by the group. The delicate and vulnerable hybrid vitroplantlets were micrografted ex vitro onto the rootstock seedling as has been elaborated later in this paper. Breeding for special objectives Dwarfness - for orchard management and fruit quality: Owing to the obvious benefits of comparatively dwarf trees for orchard management and fruit quality, attempts were focused on obtaining hybrids with a dwarf tree framework. Breeding for dwarfness is important in mango, since, a consistent dwarfing effect of any rootstock has not been established to date. Indian cultivars found to be useful as a source of imparting dwarfness include ‘Kerala Dwarf’, ‘Amrapali’, ‘Janardhan Pasand’ and ‘Nileshwar Dwarf’ (Singh, 1990; Iyer and Subramanyam, 1986). Controlled crossing was effected initially for three consecutive years by the authors, using following mango varieties identified on the basis of breeding priority: S. No. Cross-combination 1. ‘Amrapali’ X ‘Kerala Dwarf’ – (AKD) 2. ‘Alphonso’ X ‘Kerala Dwarf’ – (ALK) 3. ‘Arka Anmol’ X ‘Kerala Dwarf’ – (AnKD) 4. Open-pollinated Totapuri (OpTp) Young fruitlets from these crosses involving monoembryonate cultivars were harvested at 4-6 weeks post-pollination and excised embryos cultured in vitro. In AKD, 67.24% (39 out of 58) excised embryos cultured on defined media formed normal plantlets in vitro, while, in OpTp 15 out of 20 (75%) embryos cultured formed plantlets. Season I data on in vitro culture and regeneration response is presented in Table 2. Type of initial response in ‘Alphonso’ J. Hortl. Sci. Vol. 8(1):1-20, 2013 Hybrid embryo rescue in horticultural crops 6 X ‘Kerala Dwarf’ (ALK) is shown in Table 3 which draws attention to the fact that nearly 93% of fruitlets from a controlled cross may be lost to unforeseen circumstances. Table 4 shows the same cross in Year II up to transfer of hybrid plantlets into mini-pots. Table 2: Data on controlled crossing in ‘Alphonso’ X ‘Kerala Dwarf’ in Year I Parameter Result Total no. of panicles used for crossing 487 Total no. of flowers used as female parent 2646 Fruit set (No.) 208 % fruit set 7.86% No. of fruits lost to leaf hopper infestation/ 193 (92.8%) sooty mold No. of exciseable hybrid embryos 15 Ratio of panicle to fruit set 1:0.42 Ratio of flowers crossed to fruit set 176.4:1 Table 3: Data on culture and regeneration response of hybrid embryos in mango in Season I Parameter AnKD* OpTp** No. % No. % a. No. of embryos 243 NA 232 NA inoculated b. No. of embryos 185 76.13 170 73.28 forming plantlets in vitro c. No. of plants 206 84.77 134 57.76 transferred from culture vessel into minipot d. No. of potted plants 48 25.95 24 14.12 surviving primary transplantation e. No. of potted plants 10 4.11 2 0.86 surviving secondary transplantation *AnKD: ‘Arka Anmol’ X ‘Kerala Dwarf’; **OpTp: Open-pollinated ‘Totapuri’; NA: Not Applicable Table 4: Data on controlled crossing and hybrid plants transferred ex vitro in ‘Alphonso’ X ‘Kerala Dwarf ’ cross in Year II Parameter Result No. of panicles used for crossing 221 No. of flowers used as female parent 857 Average no. of flowers crossed/panicle 3.878 Initial fruit-set (no.) 57 No. of fruits lost to fruit drop 7 % Fruit-set 6.65% No. of fruits used for embryo excision 50 No. of cultures contaminated in vitrovent vessels 7 No. of cultures showing no response 4 No. of plants transferred to mini-pots 39 Comparative analysis of reproduction parameters for three years on controlled crossing in this cross is presented in Table 5. Table 5. Comparative analysis of reproduction parameters for three years on controlled crossing in ‘Alphonso’ X ‘Kerala Dwarf’ Parameter Year I Year II Year III No. of panicles used for 496 221 487 crossing No. of flowers used as 3531 857 2646 female parent Fruit-set (no.) 164 57 208 % Fruit-set 4.64% 6.65% 7.86% Ratio of flowers crossed to 22:1 15:1 13:1 fruit-set No. of fruits used for embryo 109 50 15 excision A comparison was made between survival rate of hybrids of ‘Amrapali’ X ‘Kerala Dwarf’ and open-pollinated ‘Totapuri’ (Table 6). Table 6: Comparative survival rate of plantlets at different stages of development in vitro and ex vitro Particulars ‘Amrapali’ X Open-pollinated ‘Kerala Dwarf’ ‘Totapuri’ % of embryos forming 67.24 75.1 plantlets in vitro % Survival of plants from 56.40 51.0 test-tube to pot Success % in survival from 100.00 80.3 pot stage to field Although the rate of ‘normal plantlet’ formation in vitro in AKD during the first season (Fig.1) was very high (67.24%), mortality from the stage of culture vessel to field- planting (through mini-pots and later, into large pots) was very high too (48.7%), ie., only 20 hybrid plantlets out of 39 AKD survived to the field-planting stage (survival rate 51.3%). In OpTp, only 20% of the plantlets survived field- planting. In the following season, in ALK, 88 hybrid embryos out of a total of 109 from the controlled cross formed plantlets in vitro (ie., 80.73%). Of the 88 hybrid plantlets transferred extra vitrum, only 2 survived to the field-establishment stage (ie., 2.27% survival rate). In the same year (1999), 105 embryos of OpTp formed plantlets in vitro out of 127 embryos cultured (ie., 82.68%). Four OpTp plantlets survived upto the final field-establishment stage (ie., 3.81% survival rate). In the third season, in AnKD, 244 hybrid embryos were excised and cultured in vitro of which 177 formed normal plantlets (72.5%). In OpTp, of the 232 embryos, 166 formed plantlets in vitro ie., 79.55%. Plantlets were transplanted into soil in minipots and subsequently, into large pots. In ‘Amrapali’ x ‘Kerala’ Dwarf (AKD), 69.23% cultures responded, with shoot and root induction. Upon J. Hortl. Sci. Vol. 8(1):1-20, 2013 Leela Sahijram et al 7 normal plantlet development, the hybrid vitroplants were transplanted extra vitrum in a phased manner into mini- pots. Eight weeks later, these hybrid plants were transferred to larger pots and finally, into the field. Field-established plants showed overall survival percentage of 34.4% in AKD and 20.0% in OpTp. In the controlled cross Alphonso X Kerala Dwarf (ALK) and open-pollinated Totapuri (OpTp), induction of rooting was achieved in 70.9% and 72.4% cultures, respectively, in Season II. Performance of ALK and OpTp was comparable (77.8 and 77.5%, respectively) on medium containing 1500 mg/l casein hydrolysate (CH) and 6% sucrose (w/v). OpTp responded best (87.5%) on medium containing 1000 mg/l CH + 3% sucrose (w/v). Cultures showing both shoot and root induction were transplanted extra vitrum into large, polyvinyl pots. In all, 80.7% cultures of ALK and 82.7% of OpTp were transferred to soil. After 4 – 5 months, the plants were transferred to large earthen pots. Finally, an overall survival percentage of 4.54% and 3.81%, respectively, of ALK and OpTp plants was obtained. In season III (year 2000), 84.77% of AnKD cultures and 57.76% of OpTp cultures developed into plantlets in vitro. Overall percentage survival in AnKD was found to be 4.11, and 0.86 in OpTp. Controlled crosses between the parental lines ‘Alphonso’ X ‘Kerala Dwarf’ (ALK) were repeated owing to poor survival in the earlier cross. Embryo excision was carried out at 6-8 weeks post-pollination (WPP). Of these, 78% of vitroplants were transferred to mini-pots (Leela Sahijram et al, 1999, 2009). Direct crossing between these parental lines (ALK) was repeated. Zygotic embryos here too were excised at 6 – 8 weeks post-pollination. In all, during five seasons of explanting, various controlled crosses were made with ‘Kerala Dwarf’ as the male parent. Cultivars Amrapali, Alphonso, Arka Anmol and Ratnagiri Alphonso were used as the female parent. Open- pollinated Totapuri, Alphonso and Ratnagiri Alphonso were also used in the study. Zygotic embryos were excised aseptically at 5 to 8 weeks post-pollination and inoculated onto modified semisolid MS medium with complex growth addenda. Factors detrimental to initial embryo growth and development were found to be fungal/ bacterial contamination, excessive exudation of phenolics into the medium, and failure of the embryo to respond. A protocol was earlier also worked out for the cross ‘Mulgoa’ X ‘Neelum’. Using immature zygotic embryos as starter explants, Leela Sahijram et al (1996) could also induce somatic embryogenesis in mango embryos / nucellus and elucidate factors influencing the same. These plantlets did not survive beyond four months of ex vitro transfer. Ex vitro shoot-tip grafting (Ex vitro STG) In the earlier seasons, transfer of vitroplants to soil resulted in very high mortality despite a healthy status of these plants at transplanting. Hence, a novel technique of ex vitro shoot-tip grafting (ex vitro STG) was devised by the authors (Leela Sahijram, 2009; Anon., 2002) using ‘Totapuri’ as the rootstock. The hybrids were grown to maturity and evaluated. Further multiplication of these hybrids was done onvarious rootstocks. ‘Totapuri’ (rootstock) stones were germinated in the traditional manner in seed pans on a sand medium. Shoot apices of the hybrid plantlets were excised and grafted ex vitro onto the rootstock using the epicotyl grafting technique. Diameter of the scion shoot apex at the cut surface was 2 - 4 mm. Graft-union established successfully in 3 weeks Table 7. Comparison between cross-combinations in crosses effected during five years Season Cross No. of % embryos % cultures Survival % from combination* embryos forming plantlets surviving transplant mini-pot stage excised for in vitro culture into mini-pot to larger pots/field 1998 AKD 58 67.24 56.80 34.48 O p T p 20 75.00 50.00 20.00 1999 ALK 109 80.73 80.73 1.83 O p T p 127 82.67 82.67 2.36 2000 AnKD 243 76.13 84.77 4.11 O p T p 232 73.28 57.76 0.86 2001 ALK 50 70.80 78.00 0.00 2002 ALK 15 73.33 60.00 66.70 OpRA 63 63.49 45.00 72.22 O p T p 23 60.86 92.85 92.31 OpA 17 35.29 66.70 100.00 *AKD= Amrapali x Kerala Dwarf; OpTp= Open-pollinated ‘Totapuri’; ALK= ‘Alphonso’ X ‘Kerala Dwarf’; AnKD= ‘Arka Anmol’ X ‘Kerala Dwarf’; OpRA= Open-pollinated Rathnagiri Alphonso; OpA= Open-pollinated Alphonso J. Hortl. Sci. Vol. 8(1):1-20, 2013 Hybrid embryo rescue in horticultural crops 8 Fig 1. Hybrid embryo rescue in mango cross ‘Amrapali’ X ‘Kerala Dwarf’: A. In vitro growth of embryo; B. Secondary root induction; C. Hybrid vitroplantlets ready for transfer; D. Etiolated seedling of cv. Totapuri for receipt of graft; E. Ex vitro shoot-tip grafting; F. Graft-union secured: G. Polybagged graft; H. Development in minipots; I. Field-establishment of hybrid; J. Flowering hybrid; K. Fruit induction; L. Mature fruits A B C D E F G H I J K L J. Hortl. Sci. Vol. 8(1):1-20, 2013 Leela Sahijram et al 9 and, at the end of 8 weeks, the scions showed vigorous growth and development. Overall graft success rate was 79.54%. Ex vitro STG was carried out using vitroplantlets of Alphonso X Kerala Dwarf, open-pollinated Alphonso (OpA), Ratnagiri Alphonso (OpRA) and Totapuri (OpTp) with a success rate of 66.7%, 100%, 72.2% and 92.31%, respectively. These grafts were transferred to polybags of size 9" height X 7" diameter containing sand, soil and vermicompost @ 1:1:1 at 10-12 weeks. Further, the grafts were transferred to larger pots containing sand, soil and farmyard manure @ 1:1:1, at 6 months. These grafts were further field-planted and hybrids evaluated for desirable traits (Fig. 1). Results of crosses effected during five years are compared and summarized in Table 7. Molecular studies on putative mango hybrids obtained through embryo culture Morphological parameters of the two parents in a cross and their hybrid progeny can be compared to look for clear-cut differences. Morphological traits, however, are subject to environmental influences and can be misleading. Besides, these lack power of resolution to identify hybrids at juvenile stages, for which it becomes necessary to wait until plant maturity (Debbarama et al, 2013). Distant crosses rescued through embryo rescue can be confirmed for hybridity using molecular markers such as RAPD, AFLP, etc. Therefore, testing of hybridity using molecular markers is advantageous, as, it obviates these hindrances. Molecular markers Mini- and micro-satellite DNA markers are highly polymorphic in many species due to a variable number of tandem repeats. DNA fingerprints obtained by hybridization of mini- and micro-satellite probes with genomic DNA were shown to be useful for cultivar identification. DNA fingerprint information for identification of nucellar and zygotic seedlings was attempted. For this purpose, DNA was isolated using CTAB method from fully mature leaves of 6 month old seedlings (stones with single-plants, as well as polyembryonic seedlings) and from field-grown mother trees. DNA samples were quantified using spectrophotometer and confirmed for presence of DNA using agarose gel electrophoresis. To multiply Jeffrey’s minisatellite probe (33.6 plasmid DNA was used as the probe), the plasmid was incorporated into DH5a E. coli competent cells and cultured on LB Agar medium with Ampicillin @ 50mg/ml. Single colonies from the transformed ones were isolated and inoculated onto the same medium. After adequate growth of the culture, the plasmid was isolated and checked for presence of minisatellite 33.6 (700 bp DNA) on 0.8% agarose gel, using restriction enzymes Bam H1 and EcoR1. Further development of the non-radioactive labelled probe (33.6 minisatellite), restriction digestion of the plant genomic DNA and Southern transfer/hybridization was worked out. PCR-amplification using ISSR primers ISSR primers UBC 814, 835, 841, 844, 848, 868, 873, 881, 898, 901 were used. The reaction mixture (20µl) contained 10mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl 2 , 0.2 mM mixed dNTP, 1 mM primer, 0.5 unit Taq polymerase and 25 ng/µl of genomic DNA. DNA amplification was obtained through 40 cycles (92o C for 1 min, 42o C for 1 min 30 sec and 72o C for 1min 30 sec), followed by extension at 72o C for 8 min. Of the ten ISSR primers used, only six (UBC 835, 841, 848, 868, 873, 898) showed amplification. Bands amplified in the hybrids were similar to that in the parents suggesting that these were true hybrids. Forward and reverse primers, viz., 1F1R, 5F5R, 6F6R and 9F9R were also used, of which 1F1R, 6F6R and 9F9R showed amplification. Seedless grapes The second food crop to be fully sequenced genomically after rice is the grape. Improvement in grape is slow and time-consuming since the crop is heterozygous and exhibits pronounced inbreeding depression (Leela Sahijram, 2011). Thompson Seedless is the ruling variety of grape in India and other parts of the world, but, is susceptible to Downy mildew disease caused by the fungal pathogen Plasmopara viticola. Obtaining a downy mildew resistant Thompson Seedless would be a boon to the global viticulture industry, resulting in massive savings in pesticide-use while providing a cleaner, safer environment. Seedless grapes are stenospermocarpic, in that, these form embryos successfully, but the embryo does not develop, and aborts due to inability of the ovular wall to expand. These embryos can, however, be rescued before they abort by initially culturing the whole ovule; after about eight weeks, the developing embryo can be dissected out from the ovule and grown on a different medium. This is termed ‘sequential embryo culture’. Ramming (1990) pioneered hybrid embryo rescue in seedless grape breeding. Sequential hybrid embryo rescue was carried out by the authors using ‘Thompson Seedless’ as the maternal parent and 15 different pollen-donor parents J. Hortl. Sci. Vol. 8(1):1-20, 2013 Hybrid embryo rescue in horticultural crops 10 Fig 2. Sequential embryo culture in Thompson Seedless grape breeding: A. Grape bunch at six weeks post-pollination with seed traces; B. Enlarged longitudinal section of fruitlet; C. In ovulo embryo culture; D. Excised embryo under high magnification; E. Germination of hybrid embryos; F. Transfer of plantlets to liquid medium; G. Hybrids transplanted into polybags; H. Plants ready for shifting to glasshouse; I. Acclimatization in glasshouse; J. Field-establishment of hybrids A B C D E F G H I J J. Hortl. Sci. Vol. 8(1):1-20, 2013 Leela Sahijram et al 11 (Tables 8) variously resistant to Downy mildew disease caused by the Deuteromycetan fungal pathogen (Leela Sahijram, 2005), as mentioned above. Open-pollinated ‘Thompson Seedless’ was also used by the authors who used a 2-step in vitro procedure: in ovulo embryo culture followed by ex ovulo embryo culture (sequential embryo transfer) to rescue immature embryos of these conventionally intractable crosses (Kanamadi et al, 1996, 1999a,b,c; Leela Sahijram and Kanamadi, 2004; Leela Sahijram et al, 2005a,b; Anon., 2005). In our studies (Leela Sahijram, 2004b), we found that pollen-donor parent overwhelmingly determined the outcome of crossing and success rate thereof (Table 9). From the directed crosses, of the 1500 hybrids generated and grown in the glasshouse, about 700 were transferred to the field (Fig. 2), established to maturity and evaluated for downy mildew resistance while retaining desirable pomological qualities of the seed-parent. In some crosses involving Seedless X Seedless parents, the authors have demonstrated through histological studies that the zygote does not develop beyond first cell division (i.e., 2-cell stage). Table 9. Comprehensive data on crossing ‘Thompson Seedless’ (female parent) and 15 pollen-donor parents resistant to Downy mildew Sl. Male Parent No. of Total No. of ovules obtained Total Total Total No. of No. panicles no.of 6-11 weeks post-pollination no. of no. of no. of fully used buds berries ovules apparently developed used 6 7 8 9 10 dissected recovered viable plants in a cross embryos regenerated 1 V. tilifolia 5 700 31 53 - - 81 289 165 28 24 2 Madras Field 4 1070 21 41 - 24 48 303 134 38* 54 Court 3 F. veltiner 4 630 8 26 - 76 0 276 110 36* 39 4 V. candicans 6 1230 0 0 - 29 10 446 39 2* 3 5 S. V. 12375 1 80 na - - - - 0 0 0 0 6 S. V. 23501 2 230 0 5 - - - 123 5 0 2 7 S.V. 18315 4 460 55 103 - - 14 247 172 20 11 8 S. V. 12309 3 360 10 30 18 - - 146 58 21* 27 9 Catawaba 3 360 12 1 - - 47 192 60 7* 18 10 James 3 210 23 51 43 - - 195 117 16 15 11 Riparia X 3 180 - 114 - 20 - 161 134 21 20 Rupestris 12 S. V. 12364 6 415 41 44 104 - - 269 189 35* 48 13 V. assamica 3 210 19 - 92 - - 191 111 20* 26 14 S. V. 18402 3 220 17 - 34 - - 225 51 8 7 15 V. lanata 1 100 - - - - 8 44 8 3 0 Total 51 6455 3107 1353 255* 294 OpTS# 52 37 11 8 Total no. of crosses = 15 Source: Leela Sahijram et al, 2005b Average no. of buds used per panicle = 127 Ovules harvested at an age ranging from 6-11 weeks post-pollination *No. of apparently viable embryos but some of the apparently non-viable embryos also developed into plantlets #Open-pollinated Thompson Seedless Table 8. Data for Year I on crossing ‘Thompson Seedless’ (female parent) with 14 Downy mildew resistant pollen-donor parents, and, open-pollinated ‘Thompson Seedless’ Sl. Pollen-donor Total no. No. of % hybrid No. parent used of ovules fully plants cultured developed recovered from hybrid the cross plantlets recovered 1 Vitis tilifolia 165 28 16.97 2 ‘Madras Field Court’ 134 57 42.54 3 Fruehort veltliner 110 48 43.64 4 Vitis candicans 39 3 7.69 5 ‘SV 23501’ 5 0 0.00 6 ‘SV 18315’ 172 14 8.14 7 ‘SV 12309’ 58 33 56.90 8 ‘Catawaba’ 60 23 38.33 9 ‘James’ 117 19 16.241 10 ‘Riparia X Rupestris’ 134 25 18.66 11 ‘SV 12364’ 189 64 33.86 12 Vitis assamica 111 26 23.42 13 ‘SV 18402’ 51 11 21.57 14 Vitis lanata 8 0 0.00 15 Open-pollinated 37 13 35.14 Thompson Seedless Total 1390 364 26.19 J. Hortl. Sci. Vol. 8(1):1-20, 2013 Hybrid embryo rescue in horticultural crops 12 Bharathy et al (2003, 2005) in their study on ‘Thompson Seedless’, ‘Flame Seedless’ and eight other varieties of grape found embryo recovery to increase significantly with application of benzyladenine - a cytokinin - at pre-flowering and flowering stages. Maximum embryo recovery (47.57%) was obtained with ‘Thompson Seedless’ (TS) × ‘Concord’, followed by TS × SV18402 (29.75%). Murthy et al (2006) achieved field-establishment and screened embryo-rescued hybrid seedlings of grape with special reference to downy mildew. Molecular markers: Leela Sahijram and Raghavendra (2005), Raghavendra et al (2006) and Leela Sahijram and Bhaskara Reddy (2009) from the authors’ laboratory carried out molecular studies using ISSR markers in seedless grape hybrids generated by the authors and confirmed their hybridity. Papaya Leela Sahijram and Doreswamy (1993) tested the efficacy of using placental cultures for rescuing hybrid embryos from controlled crosses involving Carica papaya and Vasconcellia cauliflora. They were able to induce transient expression of anthocyanin gene (inky-blue in colour) in placental cultures of hybrid papaya. Intergeneric and interspecific crosses were used for hybrid embryo rescue and hybrid plants were recovered. Vitroplantlets were transplanted into soil ex vitro (Fig. 3). Seedless lime Prasad et al (1996) emphasized the importance of embryo rescue in improving seedless lime quality. Controlled crossing between ‘Seedless lime’ and Acid lime resulted in immature zygotic embryos that were rescued in vitro (Fig. 4) and grown to maturity in the field. The objective here was to develop a hybrid of acid lime with resistance to citrus canker. Role of embryo culture techniques in improvement of fruit quality in seedless lime was elucidated by Prasad et al (1996). Rao et al (2011) highlighted the importance of genetic resources in citrus fruits and their exploitation in citrus improvement programmes using various in vitro techniques such as embryo rescue, nucellus culture, etc. Banana C o m m e r c i a l l y popular varieties are seedless owing to their triploid status. Protocols have been developed for rescuing excised hybrid embryos from crosses involving Musa acuminata and M. balbisiana (Fig. 5). Hybrid plants were raised to maturity in the field and evaluated (Doreswamy and Leela Sahijram, 1991, 1993; Chadha and Leela Sahijram, 2000). The hybrids were added to the germplasm pool in the Institute for further use in breeding. Olive Acebedo et al (1997) found the growth of plantlets derived from in vitro germinated embryos in the greenhouse to be normal. Thus, embryo culture can increase the efficiency and shorten the time for starting initial progeny evaluation and thereby speeding up seedling development in olive breeding programmes. VEGETABLE CROPS Wide-hybridization to a vegetable breeder and cytogeneticist is the first step in transferring genes from Fig 4. Breeding Seedless Lime X Acid Lime: Hybrid embryos rescued in vitro (Left) and potted hybrids (Right) Fig 3. Intergeneric breeding strategy in papaya: Hybrid plantlets in vitro (top) and transfer to soil (bottom) Fig 5. Rescued interspecific cross Musa acuminata X M. balbisiana: A. Excised embryo on semisolid medium; B. Germination; C. Plantlet development; D. Profuse rooting in the hybrid banana J. Hortl. Sci. Vol. 8(1):1-20, 2013 Leela Sahijram et al 13 wild species into cultivated ones. Embryo rescue technique has been successfully demonstrated in tomato, capsicum, chillies, okra and radish. In India, embryo culture work has been initiated in okra for rescuing interspecific hybrids. The following literature is by no means an exhaustive account of work done. Tomato Smith (1944) and Demirel & Seniz (1997) made observations on the possibility of utilizing embryo culture for improvement of tomato. For in vitro breeding in tomato, Aragao et al (2002) used three culture media in combination with distinct accessions, crossing generations, and periods of time after artificial pollination, and evaluated them to identify more efficient protocols to recover interspecific hybrids between Lycopersicon esculentum and L. esculentum. Bhattarai et al (2009) found germination of immature seeds to be a better alternative to culture of excised immature embryos in hastening tomato breeding programmes. Capsicum / Chilli In the state of Meghalaya in India, chilli is the third most important spice crop after ginger and turmeric. However, diseases namely, tobacco mosaic virus, root rot, tomato spotted wilt virus, etc. lead to considerable decline in yield. Capsicum chinense, C. annuum and C. frutescens were crossed with each other and embryos rescued between 27-33 days after pollination. Highest percentage of embryo growth was observed on MS medium with 0.5 mg/l GA 3 and 0.05 mg/l NAA. Hybrid plants were obtained and their hybridity confirmed using morphological as well as RAPD markers (Debbarama et al., 2013). Radish Intergeneric herbicide-resistance transfer to radish has been recently accomplished using embryo rescue (Mithila and Hall, 2012). For introgressing auxinic herbicide (Dicamba) resistance from wild mustard (Sinapis arvensis) into radish (Raphanus sativus; 2n=18), embryo regeneration and hybrid plant production was achieved involving several hundred reciprocal crosses performed between these two species. Upon altering cultural conditions and media composition, a high frequency of embryo regeneration and hybrid plant establishment was achieved. Okra As resistance is not available in cultivated species of okra [Abelmoschus esculentus (L.) Moench.], interspecific crosses were made between A. esculentus and A. moschatus (resistant wild species) to develop resistant varieties. Post-zygotic incompatibility was found to operate between the species. Crossed seeds were shriveled and non-viable. In vitro embryo rescue to overcome the incompatibility revealed that culturing 12 and 15 day old embryos of A. esculentus var. Kiran (a high yielding line) x A. moschatus and A. esculentus var. Anakomban (a landrace) x A. moschatus on MS medium supplemented with BA 0.5 mg l-1 and CW 150 ml l-1 yielded transplantable hybrids. Embryos of crosses A. moschatus x A. esculentus (i.e., reciprocal cross) turned brown by the 11th day of pollination and could not be cultured in vitro (Rajamony et al, 2006). The authors’ laboratory has initiated work on in ovulo and ex ovulo culture of okra embryos (Fig. 6) with a view to generating interspecific hybrids in the long run. In another study (Fatokun, 1987), crosses were made between members of two West African okra types, ‘Soudanien’ and ‘Guineen’. All crosses succeeded in both directions but F 1 plants that showed hybrid vigour for plant stature were partially sterile. Cytological observations of these F 1 plants revealed abnormal meiosis which resulted in production of microspores of variable size. Frequency of viable pollen (indicated by acetocarmine staining) was low in the hybrids: 35.80% (U.I.92× U.I.313) and 39.41% (1bk-1×U.I.215). Number of seeds produced per fruit was also low in the hybrids and only a few of these seeds were viable. Brinjal Incompatibility barrier was overcome in brinjal in F1 hybrids of the interspecific cross involving Solanum melongena and S. macrocarpon. Reciprocal crosses made by the breeder resulted in F2 progeny. BC1 - Backcross 1, [BC(H1 x M) Pl. 3] (Fig. 7), was rescued and has been transferred to soil for evaluation (Leela Sahijram and Padmini, unpublished data 2013). Fig 6. In ovulo embryo culture in okra J. Hortl. Sci. Vol. 8(1):1-20, 2013 Hybrid embryo rescue in horticultural crops Fig 7. Hybrid plantlet of brinjal rescued from an interspecific cross 14 Artichoke Globe artichoke breeders have two important problems: lengthy seed-to-seed cycle, and seed-borne diseases caused by fungal pathogens. Both problems can be solved by embryo rescue. Embryos were collected from five cultivars in different post-pollination stages (Cravero and Crointy, 2007). Root induction was poor, but shoot development per explant was better. Twenty days were required as the optimal time for embryo rescue. ORNAMENTAL CROPS Van Tuyl and Lim (2003) reviewed interspecific hybridization and polyploidization as tools in ornamental plant breeding. Several crops in floriculture have benefitted from in vitro interventions in breeding programmes. Some of the successes are reported herein. Rose Marchant et al (1994) used embryo culture for production of F1 hybrids in English rose. Rout et al (1999) reviewed advances made in the biotechnology of rose and Holeman (2009) outlined a simplified method of embryo culture in rose. Hybrid / zygotic embryos were rescued form controlled crosses and open-pollination, respectively, in rose cultivars. Mohapatra and Rout (2005) in their study on embryo rescue in floribunda roses rescued immature embryos from “Arunima’ and “Shocking Blue’ roses. Time required for breeding roses through conventional breeding, for example, is very long. It is occasionally hampered by premature abortion of the developing embryo, resulting in few or no viable seeds (Rout et al., 1999). Rose is highly heterozygous. Lack of germination is due to mechanical restriction of embryo-expansion by presence of a thick, hard pericarp or due to dormancy regulated by growth inhibitors present within the achene. There are also a few reports of culture of mature embryos in vitro. Embryo rescue in Rosa hybrida and English roses is also reported from elsewhere. Gudin (1994) also demonstrated the usefulness of embryo rescue in Rosa hybrida L. Lilies / Liliums In order to introduce new traits such as disease resistances, flower shape and colour from wild species into the cultivar assortment of lily, it is necessary to overcome interspecific crossing barriers. Several techniques like the cut-style method, the grafted-style method and in vitro isolated ovule pollination technique, have been developed to overcome pre-fertilization barriers. Post-fertilization barriers can be circumvented by in vitro pollination and/or rescue methods as embryo, ovary-slice and ovule culture. Using these techniques, wide interspecific lily crosses with species and cultivars from the different sections of the genus Lilium (L. longiflorum, L. henryi, L. canadense, L. pardalinum, L. concolor, L. dauricum, L. candidum, L. rubellum, L. martagon, Asiatic and Oriental hybrids) could be made (Van Tuyl et al, 2000; Van Tuyl et al, 2002) and breakthroughs achieved. Interspecific hybrids have been developed in liliums using early-stage ovule culture in vitro (Wang et al, 2009). Yuan et al (2003) demonstrated shortening of breeding cycle in spider lilies (Lycoris spp.) through embryo culture. Differential ovule development following self and cross pollination and the basis of self-sterility in Narcissus triandrus (Amaryllidaceae) was shown by Sage et al, 1999. Earlier, Hayashi et al (1986) demonstrated ovary-slice culture in Lilium formosanum Wallace and Ikeda et al in 2003 produced seedlings from ovules excised at a time when they contained zygote stage of the hybrid product and cultured them in Lilium spp. Pelargonium As a means of integration of in vitro techniques in ornamental plant breeding, Bentvelsen et al (1990) made interspecific crosses in Pelargonium by applying embryo rescue methods. Some reports of artificial hybrids are available but no evidence of natural hybridization is seen. Alstroemeria Interspecific hybridization in the genus Alstroemeria is hindered by post-fertilization barriers (Buitendijk et al, 1995). Histological analysis revealed poor endosperm development from 18 days after pollination onwards, followed by malformation and abortion of embryos. To create interspecific hybrids between Alstroemeria aurea, A. pelegrina, A. magnifica, A. inodora and A. psittacina in diallelic combinations, an ovule culture technique was developed. Influence of age of ovules, sucrose concentration of medium and temperature and light during culture were tested. Harvesting ovules before the onset of endosperm degeneration, i.e. at 14 days after pollination, cutting them into halves and culturing the micropylar halves in a rotating liquid culture medium containing 6% sucrose at 21 °C in the dark, led to successful embryo rescue. Germinated embryos were sub-cultured in vitro until rhizomes were formed, a prerequisite for successful transfer to the greenhouse. Full grown plants all showed interspecific morphological traits J. Hortl. Sci. Vol. 8(1):1-20, 2013 Leela Sahijram et al 15 and analysis of chromosome complement confirmed their hybrid nature. Diploid hybrid plants were obtained in all the 20 interspecific 2x-2x combinations. A total of 260 interspecific hybrid plants were produced. Half-ovule culture of 2x-4x and 4x-2x crosses resulted in 43 triploid hybrid plants. Because interspecific hybrids were obtained in 100% of the interspecific combinations, it is expected that the described technique can be applied to overcome post- fertilization barriers in most crosses within the genus Alstroemeria. Chunsheng and Bridgen (1996) studied the effect of genotype, culture medium and developmental stage of the embryo on in vitro responses of ovule cultures in the interspecific hybrids of Alstromeria. Cacti Embryo rescue and plant regeneration has been successfully demonstrated in cacti following interspecific crosses in the genus Hylocereus (Aroldo and Noemi, 2010). Gynogenesis has also been shown to be possible in the vine cacti Hylocereus and Selenicereus by Benega-Garcia et al (2009). Other ornamental crops In primulas, Kato et al (2001) obtained different genomic combinations in inter-section hybrids from crosses in primulas through embryo rescue. Two types of triploids with different genome combinations were generated. Interspecific hybridization in rhododendron has been shown to be possible using embryo rescue in the genus Rhododendron (Tom Eeckhaut et al, 2007). Other attempts at embryo rescue include that in Narcissus and Zinnia (Miyajima, 2006) and Ornithogalum (Josephina, 1990). Kasten and Kunert (1991) devised a method for culture of isolated immature embryos of various lupins (Lupinus species); Custers (1995) overcame interspecific crossing barriers in tulips using embryo rescue by successful direct transfer of Tulipa kaufmanniana Regel germplasm into that of T. gesneriana L. Tuber crops: Cassava Cassava is one of the most-planted tuber crops in the tropical world. Importance of cassava is growing as a food security crop in sub-Saharan Africa, where malnutrition is a menace. However, a major hindrance in fast improvement of the crop is a long generational-cycle in this crop, poor germination of the seeds, and low multiplication-rate of the stem cuttings. In vitro germination of 495 seeds from a backcross population was done. Each genotype was multiplied for sufficient planting material, hardened in the greenhouse and transplanted to the field. Percentage germination of the seeds in embryo culture was high (66%). Raising plantlets in the greenhouse was found to be useful to select healthy plants and, thus, obtain a uniform stand in the field. The genotypes were planted in a single row trial and harvested eight months after planting. Transplanted plantlets gave 98.89% establishment. Yield-related traits were significantly high compared to results from past experiments. The high percentage of plant recovery from seed through to the field, is a means of overcoming some problems associated with traditional methods of cassava breeding through direct seed planting, to generate planting material (Akinbo et al, 2010). BASIC STUDIES CARRIED OUT USING EMBRYO CULTURE: In addition to applied uses of embryo culture, the procedure is useful in basic studies. Growing embryos outside the ovule (ex ovulo) is an excellent way to study nutrition and metabolism of the embryo at various stages of its development. The technique can also be used to examine growth requirements of embryos, effects of phytohormones and environmental conditions on zygotic embryogenesis, and the regeneration potential of whole embryos or their segments (Bridgen, 1994). Embryo culture can be used to localize sites of germination promoters and inhibitors, for studies on embryogenesis, and for cryopreservation. Embryo culture is useful in understanding precocious germination. Studies have shown that seed tissues play an important role in regulating development of embryos in situ. Cotyledon growth stops almost immediately upon excision of immature embryo, indicating probable cessation of cell division, as seen in cotton. An embryo is programmed to germinate even when it is very small. In situ the embryo may not germinate because it lacks the germination-mRNA. But, removal of the seed from its environment activates the required machinery of embryo cells to synthesize the information necessary for germination. It has been shown in cotton that germination-specific mRNA is normally transcribed when cotyledons are about 3/5th their final size. However, to check precocious germination that may lead to vivipary (a lethal development), translation of mRNA is prevented by simultaneous appearance of abscisic acid (ABA) in the seed. Embryo excised at this stage undergoes precocious germination which suggests, that, ABA is contained in tissues surrounding the embryo rather than J. Hortl. Sci. Vol. 8(1):1-20, 2013 Hybrid embryo rescue in horticultural crops 16 in the embryo itself. This conclusion is also supported by the fact that exogenous application of ABA to excised embryos also prevents their precocious germination. Accumulation of ABA during late stages of seed development has been shown to occur in a number of plants. Other non-conventional approaches to breeding crop plants include: 1. Induction of haploidy through androgenesis or gynogenesis and subsequent diploidization, as a short- cut to breeding 2. Generation of triploids through endosperm culture 3. In vitro mutation breeding 4. Development of biotic/abiotic stress-resistant plants by challenging plant tissues in vitro with the specific stressant 5. Somatic hybridization using protoplast isolation, culture and fusion 6. Production of transgenics using one or more of the following technologies: (i) Microinjecting foreign DNA (ii) Use of biolistics (gene gun/ helium gun) (iii) Vector-mediated transformation CONCLUSION A very important and valuable biotechnological tool for raising hybrid progeny in intractable crosses is embryo rescue. It is most often used to rescue embryos from interspecific and intergeneric crosses and from embryos that do not fully develop naturally (as in early-ripening and seedless fruit crops where the embryo aborts). Distantly related plant species in a breeding programme (‘wide hybridization’) may result in no/aborted hybrid embryos. Seedless parents may be unable to produce seeds in a cross. Alternately, breeding may be hindered owing to heavy fruit- drop in the early stages of fruit development. In all such cases, hybrid embryo rescue is a very powerful and useful tool and an indispensable technique. The method also can be used to rescue seedless triploid embryos, produce haploids, overcome seed dormancy, or determine seed viability. It is useful in understanding embryo morphogenesis and precocious germination. As research continues with this technique, new and valuable uses will be developed to assist biotechnological breeding of plants. Embryo rescue / culture as a technique has found wide acceptance and utilization. Many interspecific and intergenetic hybrids have been successfully produced by culturing immature embryos that normally do not survive to maturity in situ. Embryo culture techniques are also used to rescue embryos from early maturing fruit varieties, to hasten maturation in some species and to overcome dormancy requirements in others. In ovulo embryo culture facilitates embryo development from the zygote stage to maturity when the ovule is cultured. A few interspecific and intergeneric hybrids have been made by first accomplishing fertilization in vitro, then culturing the ovules to maturity. Self-incompatibility in some species can be circumvented with these techniques. The potential of these may be a viable alternative to parasexual hybridization and somatic embryogenesis. REFERENCES Acebedo, M.M., Lavee, S., Linan, J. and Troncoso, A. 1997. In vitro germination of embryos for speeding up seedling development in olive breeding programmes. Sci. Hort., 69(3-4):207-215 Akinbo, O., M. Labuschagne and M. Fregene. 2010. Embryo rescue as a method to develop and multiply a backcross population of cassava (Manihot esculenta Crantz) from an interspecific cross of Manihot esculenta ssp. Flabellifolia. Afr. J. Biotechnol. 9(42):7058-7062 Anon. 2002. New Technology: Ex-vitro shoot tip grafting in hybrid vitroplants of mango obtained through embryo rescue in vitro. IIHR Newslett., Bangalore, India, XXIII Oct.-Dec. 2002, 4:2 Anon. 2005. 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