Embryo Culture and Chromosome Doubling

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Embryo Culture and Chromosome Doubling Results Crosses Green Beans and Black BeansResume of M. Kosmiatin and I. MariskaCHAPTER IPreliminary1.1 BackgroundMung bean (Vigna radiata L. Wilczek) including crops that have long been cultivated in Indonesia (Arora and Mauria 1993). Green bean cultivation is relatively easy with a small risk of failure. However, the vast increase pertanamannya only about 5% / year. Slow improvement is partly due to the difficulty of farmers obtain good quality seed and harvest time are not simultaneously (Marzuki and Soeprapto 2001). The development of a disease that has not been followed by the provision of resistant varieties were also hampered the development of green beans (Astanto and Sutarman 1993). Crosses between species plays an important role in plant breeding programs. This technique is used if desired genetic diversity not found in the cultivated species. Crosses between species can facilitate antartaksa introgressed gene (Song et al. 1997). Crosses between species is also possible to obtain hybrids with high variations, such as the mutation and adaptation expansion both abiotic and biotic environment or individuals acquire a new character combinations.A cross between green beans and black beans (Vigna mungo) has been done in India and Australia mainly to the properties of tolerance to the disease. The results indicate a miscarriage embryo crosses were quite high and the failure of germination (Jaiwal and Gulati 1995). The seeds produced are fertile F1 even partially sterile, slow cooked, and appeared several morphological characteristics that are the combination of the two parental (Gosal and Bajaj 1983).To save the embryos and increase seed germination F1 to do with culture in vitro. This technique is widely used to rescue embryos or seeds from crosses by way mengkulturkannya the appropriate growth medium (Raghavan 1986). The success of in vitro culture techniques is determined by several factors, including the condition and kinship genotypes used as elders, media formulation, and age younger seeds (embryos) were cultured. Crosses between species generally produce fertile F1 plants partially full of pure sterile, because the genomes derived from different elders, so that when the formation of meiotic cell division multivalent (Poespodarsono 1988).Consequently gametes formed a khimera chromosome (Hansen and Andersen 1998). Fertile F1 plants from crosses with different genomes can be obtained if the form amphidiploid or allotetraploid. Ampidiploid Plants can be obtained by doubling the plant genome and the genome F1 elders to be crossed, and perform somatic hybridization (protoplast fusion) of two elders.Artificial chromosome doubling is generally done by adding the compound colchicine. These compounds will inhibit the formation and activity of spindle threads during mitosis, where chromosomes in metaphase stage does not move towards the two poles but remained in the equatorial region can even be re-doubled (Strickberger 1985). Compounds colchicine can be used in both in vivo and cultured in vitro. Efficiency is achieved both are relatively the same way, but more effective in vitro culture for its treatment may be imposed at the cellular level (Husni et al. 1995).Rescue embryos with embryo culture and embryo chromosomal duplication F1 from crosses amphidiploid plant is expected to produce a normal and fertile. Furthermore, plants from crosses can be selected and tested resistance to scurvy. This study aims to get black beans tolerant accession scurvy suitable for green bean varieties crossed with Swallow, knowing the right time to save the life of the embryo and seek appropriate media formulations for mengecambahkannya, and determine the concentration of colchicine to double the chromosome F1 from crosses.
1.2. DestinationThis study aimed to gain accession to the appropriate black beans crossed with green beans, age appropriate for embryo rescue, appropriate media, as well as the concentration of colchicine to double the chromosome F1.
CHAPTER IIMaterials and Methods
The research was conducted in October 2002 until May 2004 at the tissue culture laboratory and glass room Research and Development Center for Biotechnology and Genetic Resources for Agriculture, Bogor. Plant material that is used as a cross elders are varieties of green beans swallow as elder females and black bean accession number VR-34, VR-35, and local Madura No. 19/1 as the elder male. The plant material grown in a glass room with fertilizer according to recommendations.2.1 Crosses Green Beans and Black BeansCrosses green beans and black beans in a room made of glass. Castration interest will digunakansebagai female elders in the afternoon (16:00 to 18:00 pm EDT) and selected before anthesis flower buds are expected to bloom next day. Pollination done after the flowers bloom at 6:00 a.m. to 8:00 pm. The pods are harvested at the age of 1, 2, and 3 weeks after pollination. Furthermore, pods stored in a refrigerator for 2 days before isolation. Data were collected for the success of pollination, pods are formed, and the pods that fall. Pollination success is characterized by withering of flowers will be pollinated and the emergence of peas.2.2 embryo cultureEmbryos to be cultured isolated from the pods are stored in the refrigerator. Before the embryo is isolated, then sterilized pods isolated embryos by opening a nut shell. Selanjutnyaembrio grown / cultured on germination medium. Embryo ages 1, 2, and 3 weeks after pollination were cultured in basic medium modified Knudson and Knudson added with BA 1 mg / l. By contrast, embryos cultured on MS medium supplemented with IAA 0.01 mg / l and kinetin 0.1 mg / l (Gosal and Bajaj 1983). Observations were made of the percentage of embryos that germinate, germination time, as well as the appearance of sprouts and cultured in vitro.2.3 Doubling the chromosomeChromosome doubling performed on young embryos the best germination. Embryos mengkulturkannya coupled with the germination medium by adding colchicine 0; 0.05, 0.15, and 0.25%, then incubated for 1, 2, and 3 days. Embryos that have been treated with colchicine subcultured on the media recovery media Knudson BA + 1 mg / l were added with 0.2% activated charcoal. Observations were made of the percentage of embryos that germinated and the number of chromosomes in the tissue treated with colchicine.

CHAPTER IIIResults and Discussion3.1. A cross between green beans and black beansThe success of interspecific crosses is determined by the closeness of relationships are. The second kinship closer elders will increase the success of crosses, whereas the more distant relationships that will minimize crossover success. The use of three varieties of black beans is intended to assess the most suitable varieties were crossed reciprocally with green beans and produce seeds that can germinate in order to obtain F1 plants were fertile. The three accessions used black beans have a high resistance to the disease scurvy.The success of hybrid varieties of green beans swallow black beans with three numbers VR-35, VR-34, and local Madura No. 19/1 is generally low (Table 1). This is indicated by the percentage of pods that can be harvested (age 3 weeks after pollination) were only 21.7 to 51.3%. The percentage of successful pollination is high with a range from 72.5 to 90.0%. This suggests that kinship green beans and black beans close enough. Genetic barrier less of an effect before fertilization, but significant genetic bottleneck after fertilization (postzygotic barriers). It is characterized by high young pods that fall to the 21st day after pollination, reaching 76.5%. Autumn pods occurred since the first day to 3 weeks after pollination, even seed pods are also able to fall. In the fall of pods, embryos are generally cultured seam so it can not. Death of young pods can be caused by an incompatibility after fertilization, because the endosperm fails to develop so that it can not support the growth and development of the embryo. Endosperm growth failure can be caused by a low rate of cell division or even halted, resulting in degradation of the already established jaringanendosperma (Hadley danOpenshaw 1980). In beans, growthembryo depends on the endosperm assource of nutrients.Pod formation highest percentage of success (51.3%) was obtained from a cross of green beans with black beans locally Madura No.. 19/1. Young pods fall also the lowest (32.1%) compared to crosses with VR-34 and VR-35. Sightings of pods from crosses was less well than other crosses an even number of seeds in each pod shriveled most.The best quality of peas produced from crosses Swallow the VR-35. Pods can be seeded by the number of seeds shriveled little or almost nothing, good quality seeds with cotyledons were formed. However, cross Swallow with VR-35 hard to do with the success of crossbreeding to form pods of only 21.7%, the embryos are killed fairly high (76.5%), but the success of polinasinya highest (90%).Establishment of seed varies from normal to abnormal (no maximal seed filling) so shriveled seed or ovule contains only the swell (not fertilized), which eventually fall. Skin color is a hybrid seed followed elder seed coat color is green brownish green beans. Embryos were isolated from the pods formed with skin peeling seeds, then planted on germination medium. Embryos were isolated have qualities that range from normal (embriodengan endosperm-cotyledon) to abnormal (seeds that do not have the seed embryo or vacuum).According to Miyazaki et al. (1984), the green bean seeds from crosses (female elders) with black beans (elder male) form normal to abnormal, sometimes seeds burst with prominent cotyledons or seed filling (cotyledons) was not optimal so that it looks wrinkled. The results also indicate that hybrid green beans and black beans have a relatively close kinship as to produce F1 crosses germinated. However, the proximity of the three accessions / black bean varieties used varies, where the accession VR-35 has the closest relationship with green beans compared accession VR-34 and local Madura No. 19/1.3.2 embryo culture3.2.1. Effect of age pods after pollinationIn cultured embryos from crosses between species, age of the embryo when cultured greatly affect the success of the cross, given the embryos died very high and it is not foreseeable. In this study, miscarriage pods / embryo lasts from the first day until day 21 after pollination. On the other hand, planting very young embryo facing technical problems difficult to isolate embryos from pods because the size of the embryo is very small. Moreover, the very young embryo implantation requires a more complex media formulations. The use of the more mature embryos will facilitate isolation and media formulation used is simple, but the embryos died (embryos do not germinate) will increase. In this study, embryos harvested after 1, 2, and 3 weeks after pollination for embryo cooking green beans ranged between 4-6 weeks, while the black beans 6-9 weeks. Expected until the age of 3 weeks after pollination, the embryo has not fallen yet mature enough to germinate and easily isolated. All embryos from crosses of green beans and black beans can germinate on the medium used (Table 2). Germination percentage tended to increase with age of embryos cultured. Increasing age also accelerate embryo germination time because the embryo is more mature.Percentage germination of three numbers from crosses of black beans at the same embryonic age diverse. Best germination percentage (55.13%) were obtained from embryos aged 3 weeks of elder male black bean VR-35, with an average time of 5.4 days of germination after planting. Germination occurs in almost 3 weeks old embryo with male elders black bean VR-34, and the embryos were successfully germinated takes 29 days after planting. Seeds from crosses with male elders VR-34 generally have broken skin due to the rapid growth of cotyledons thus inhibiting the growth of the embryo axis. This suggests that embryos with perfect conditions (embryo hampered by the cotyledons) take longer to germinate. Similar results were shown by the age of 1 week embryos from crosses with black beans and local VR-35 Madura No. 19/1, respectively 31.4 and 30.5 days. Crosses with elder male black bean VR-35 produces a fairly good germination at all ages embryo and media used.The use of the older embryos will increase the percentage of embryo germination and speed of germination time. It is appropriate denganPierik (1987) which states that the mature embryo culture is simpler / easier than the young embryo culture. At the age of 3 weeks after pollination, embryo fairly good condition with a perfect cotyledons. Although some embryos had large cotyledons that seed coat kinda broke, it does not interfere with germination. With near-perfect condition embryo, the embryo does not require a long time to germinate sprouts median time 5.4 days after planting.The success of germination of embryos younger embryos only 9.52% for the age of 1 week and 21.35% for the embryonic age of 2 weeks after pollination. Decrease the percentage of embryo germination followed by an increase in the average time of germination, ie 31.4 days for the embryo 1 week and 16.7 days for 2 weeks after pollination embryo. Embryo age 1 week after pollination rather difficult isolated because of its size only + 2 mm including the seed coat and the embryo is still a liquid. At the age of 2 weeks the embryo is visible, but the size is still small kotiledonnya. Embryos aged 1 and 2 weeks after pollination may require a richer media formulations or more complex to enhance germination.In crosses with the male elders of the black bean VR-34, the increase in the age of the embryos had lower germination percentage, even at the embryonic age of 3 weeks, the average germination under 1%. At the age of 3 weeks, the skin has been broken and the seed cotyledons grow very rapidly thus inhibiting the growth of the embryo axis. Something like this also happened on Solanum interspecific crosses, in which the growth of the embryo endosperm menggungguli hybrid embryos died so young (Handayani 1995).In younger embryos, cotyledon growth has not inhibited the growth of embryo axis so that the percentage of germination 22.27% (embryo 1 week) and 22.43% (embryo 2 weeks). This shows that the determination of the age of the embryo to be germinated should consider factors miscarried embryos and kinship elders used. Older embryo culture is relatively easy with a simple media formulation, but can not guarantee success for germination in vitro embryos from crosses. In crosses with the male elders of the local black beans No. Madura. 19/1, germination of embryos tend to resemble embryos from crosses with the VR35. However, growth in the local Madura No. embryo. 19/1 less than 3 weeks with an average germination time is longer than the VR-35. This shows the germination of embryos from Madura local male elders may increase when cultured at an older age. The low germination may also show discrepancies between varieties of green beans with black beans Swallow local Madura.3.2.2Pengaruh germination mediaIn embryo culture, in vitro germination success is also determined by the composition of media and growth regulators were added to the media to replace the role of the endosperm. Complete germination of embryos usually do not require complex media formulations, even in some plant species, the embryo can be grown in the basic medium without growth regulators, such as in embryos from crosses S. khasianum and S. capsicoides (Handayani 1995).Results showed that the embryos from crosses between green beans and black beans can germinate on all media used, whether a simple media (medium base Knudson), a richer media (MS) and media enriched with plant growth regulators. In general, the addition of plant growth regulators BA in basic media can increase the percentage of germination. In younger embryos, addition of BA 1 mg / l may increase the germination percentage better than without the addition of BA. In crosses with the male elders of the black bean VR-35, the best germination percentage (90%) obtained at Knudson medium + BA 1 mg / l for embryonic age 3 weeks with an average time of 3.6 days to germinate. High germination percentage (81%) were also obtained from the embryonic age of 1 week of elder male black bean VR-34 were cultured in medium modification Knudson + BA 1 mg / l. However, germination time is much longer (19.7 days). Media Knudson turned out pretty good basis for mengecambahkan embryos from crosses between species, except for the younger embryos with male elders and local VR-35 Madura No. 19/1.This is consistent with the results of Mariska et al. (1998) in embryos from crosses between species vanilla. Using MS medium diluted 1/2 times better than a full MS. Salt content diluted MS medium macro is similar to the salt medium macro Knudson. The addition of BA to the germination medium was also successful in F1 embryo germination of wild vanilla and vanilla cultivation with medium 1/2 MS + BA 1 mg / l (Mariska et al. 1998), F1 embryos Vigna unguiculata and V. vexillata on medium MS + BA 1 mg / l adenine sulfate + + casein hydrolyzate (Gomathinayagami et al. 1997), F1 embryos Glycine max and G. canescens on B5 medium + 1 μM BA + 0.1 μM NAA (Bodanese-Zanettini et al. 1996), and F1 embryos C. arietinum and C. pinnatifidum on B5 medium + BA 2 mg / l + IAA (Badami et al. 1997). Germination of embryos younger (age 1 and 2 weeks) of elder male black bean VR-35 and local Madura No. 19/1 better on the medium MS + IAA and kinetin.Growing shoots showed high diversity (Fig. 1). Shoots were then sub-cultured on MS or B5 media to propagate clonally. However, the shoots can not be doubled, both adventitious shoots and aksilar. Generally shoots showing symptoms of callus formation at the base of the stem, the leaves fall and wither and the roots are well established. Withering and cause leaf drop subculture shoots must be done quickly, within 2 weeks after planting the leaves begin to wither and eventually fall. Withering also occurs in shoots shoots were subcultured. To reduce the leaves fall and wither, try added arginine and glutamine and AgNO3 at low concentrations, but it inhibited the growth of shoots and akhirnyamati. Try root formation was induced by adding auxin (IAA, IBA, and NAA) concentrations are low, but the addition of growth regulators is precisely induce callus formation. Clonal propagation of F1 from crosses on green beans and black beans are not increasing the number of buds, but the buds actually decrease as a result of withering and dying because of frequent contamination subcultured shoots. Shoots were successfully established roots with the number of leaves over two acclimatized in a mixture of soil and compost medium. However, plantlets generally only survive for up to 2 weeks, and some plants that survive up to 12 weeks are not flowering. This suggests a hybrid is sterile, because of the mismatch chromosome antartetua that unpaired chromosomes.3.3 Doubling the chromosomeOne effort to overcome the sterility of a hybrid is to double the chromosomes are made as early as possible. Doubling at an early stage is intended to increase the chances of getting plants ampidiploid (Poespodarsono 1988). Chromosome doubling in vitro can be performed on plant material is still very young, even at the cellular level (Husni et al. 1995).In the in vitro chromosome doubling, embryos from crosses between green beans and black bean VR-35 gave the highest germination percentage. Doubling performed with cultured embryos aged 2-3 weeks on germination medium supplemented with colchicine. Results showed that the embryos failed to germinate for each replication duplication. The embryo is not dead, but failed to form buds while plumula radikel does not extend but only have swelling. Dapattumbuh first leaf and green but only lasted a few days and then fall. Cotyledons and radikel enlarged and form a callus, but the callus can not regenerate.The problem is difficult to overcome pengkalusan although I've tried to add antiauksin into the medium. In some treatments, callus formed embryogenic callus resembles, but is difficult to regenerate callus forming somatic seed or seed is successfully formed somatic imperfect. Chromosome analysis methods is difficult because callus squash grow over the dots grow, both shoot and root tip meristem. The number of chromosomes callus can not be analyzed because of the difficulty determining the metaphase stage of cell division callus cells by a very active and fast.Doubling the embryo was repeated by replacing the recovery media after colchicine treatment. Restoration done with embryos cultured on germination medium was given activated charcoal to reduce the residual 0.2% colchicine were carried away. Activated charcoal can bind to toxins released by the explants. Subcultures have reduced pengkalusan even some buds to grow (Figure 2). Shoots successfully obtained from all treatment colchicine concentrations 0.05, 0.15, and 0.25% and incubated for 2 days (Table 3). Most buds obtained from concentrations of 0.15% colchicine. Chromosome analysis performed by immersion in a solution hidroksikuinolin shorten to only 2 hours. All who live explant analyzed the number of chromosomes. Plants from crosses between species are usually sterile due to an imbalance of chromosomes during division. At frequent mitotic chromosome movement multivalent forming cells by the number of homologous chromosomes khimera little or none at all. This limitation can be overcome by artificial chromosome doubling as the colchicine treatment.The analysis showed the diversity of the number of chromosomes of the explants were successfully fixed at metaphase stage, albeit with the same colchicine treatment (Table 3). This is likely due to differences in the number of chromosomes of the F1 embryos were duplicated. Chromosome ploidy level can not be known because the embryos directly copied without prior visits chromosome number initially. The number of initial chromosomes is difficult to obtain because culture can not be propagated clonally. 0.25% colchicine treatment gave the highest number of chromosomes (72). Longer colchicine treatment also increased the number of chromosomes. Shoots obtained from cultures with the number of chromosomes 36-60.CHAPTER IVCover4.1 ConclusionElders black bean VR-35 crossed with the best match for the green beans with the results of the best F1 crosses. The embryo can germinate until the age of 3 weeks after pollination embryo, except in crosses with black bean VR-34. Germination of embryos in vitro can be done on the basis of medium Knudson. The addition of BA 1 mg / l may increase the percentage of embryo germination. Most shoots obtained from colchicine treatment of 0.15% and incubated 2 days. The number of chromosomes derived from the highest concentration of 0.25% colchicine.

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