MICROPROPAGATION OF CYMBIDIUM SPP. BY SOMATIC EMBRYOGENESIS TECHNIQUE

Cymbidium spp. was regularly produced through micropropagation by protocorm like bodies (PLBs) and multiple shoots; micropropagation takes a lot of labor, and has high cost of seedlings, energy and material. The purpose of this paper was to study the new technique of using in vitro embryogenesis culturing for microprogation to resolve the above difficulties. The method was to use PLBs as planting materials to produce somatic callus cell and embryos. Results were followed as: PLBs were cut into slices and placed on the medium for callus initiated and used as materials for embryo formation study. A fresh weight of callus were used for the experiment of about 100 mg. The medium for the initiation of embryonic callus was composed of: MS + BA (0.1 mg/l) + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (1mg/l) or 2.4D (1 mg/l) and was proliferated on the medium MS + pepton (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (1 mg/l) or 2.4D (1 mg/l). Somatic cell suspensions were initiated and proliferated on the medium consisting of MS + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (1 mg/l) + BA (0.1 mg/l). Somatic cell suspensions were differentiated to embryonic cell suspensions on the medium MS + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with BA (1 mg/l). Embryonic cell suspensions were plating and regeneration on the medium MS + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (0.1 mg/l) + BA (1 mg/l). Micropropagation of Cymbidium spp. via embryogenesis technique was set up to produce 3,800 plantlets per one liter of somatic embryogenesis suspension. UDC Classification: 57.01; DOI: http://dx.doi.org/10.12955/cbup.v6.1312


Introduction
Traditional breeding in orchids nowadays results in the problem that common for micropropagation are tissue cultures that often grow slowly and involve a lot of work to produce seedlings. Large quantities are available at high market prices for seedlings (Mamood, 1993). The somatic embryo propagation system (Evans et al, 1981) addresses the above barriers with the advantages of: rapid multiplication in the form of cells, the embryos are a differentiation with high regeneration coefficient, they are dynamic and also cost effective (Kurata and Kozai, 1992). The initial material in the embryo culture plays a role in ensuring parental traits and maintaining high regeneration rates over a long period of time (Chang and Chang, 1978). The medium for the embryo culture plays an important role in the growth and differentiation of embryonic cells (Arditii and Erent, 1993). Control of differentiation and regeneration of embryos under the influence of growth regulators has become the rule Haun and Tanaka, 2004). PLB is a particular embryo that arose in the cultivation of cytokinin-induced orchids (Park et al, 2003). A study on embryo callus growth, embryo culture, and embryo regeneration are the first barriers in somatic embryos (Jaime et al, 2006;Nayak et al, 2002). This paper investigates the rapid multiplication of Cymbidium through the somatic embryo technique.

Results and Discussion
Cultivation and Formation of Callus Formation of protocorm like body The sample culture was young leaves. The PLB formation medium: MSC + B1 (5 mg/l) + CW (10%) + pepton (1 g/l) + sucrose (20 g/l) + activated charcoal (1 g/l) with NAA supplement (0, 0.1, 1, 3 mg/l) and TDZ (0, 0.1, 1, 3 mg/l). Results of the study (Table 1): After 45 days of culture. The rate of formation of high PLB (80-90%) supplemented with NAA (0.1 mg/l) + TDZ (3 mg/l) and NAA (1 mg/l) + TDZ (3 mg/l). Growth regulators TDZ play an important role in the generation of PLB embryos. The number of PLB formation was high in NAA (0.1 mg/l) + TDZ (3 mg/l) and NAA (1 mg/l) + TDZ (3 mg/l) with 10-20 PLB/ sample.  Source: Author Source: Author Formation of embryogenic callus PLB has a slow in vitro multiplication rate. It has a large size, limited to micropropagation. It was used as raw material for the production of embryogenic callus. The PLB was cut in thin slices and placed on the culture medium to create the callus. The culture medium for callus formation: MS + BA (0.1 mg/l) + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (0.1, 0.5, 1, 2, 3 mg/l), 2.4D (0.1, 0.5, 1, 2, 3 mg/l). Results of the study (Table 2): After 45 days of culture, the culture medium containing the NAA (1 mg/l) or 2.4D (1 mg/l) stimulated embryogenic callus formation, with the formation rate of callus being low and the diameter of the callus being small. The culture process occured outside, the callus was green; otherwise, in the dark, it was ivory-white color. The ivorywhite-color embryonic callus cells were suitable for the liquid multiplication of shaking under diffused light.
Proliferation of embryogenic callus The embryonic callus from the above study was used as culture material. The weight of tissue to be cultured was 1 g/50ml culture medium. The culture medium proliferated the mass of callus: MS + BA (0.1 mg/l) + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (0.1, 0.5, 1, 2, 3 mg/l)), 2.4D (0.1, 0.5, 1, 2, 3 mg/l). The results of the study (Table 3) show that: a suitable proliferation culture medium was a medium supplemented with NAA (1mg / l) or 2.4D (1 mg/l). The ivory-white somatic cells were cultured in the dark at a rapid rate of proliferation of 4-5 times more than under diffused light after 30 culture days. Source: Author Regeneration of embryogenic callus The embryonic callus from the above study was used as culture material. The mass of tissue injected into the culture was 500mg. The culture medium for regeneration of callus was: MS + peptone (1 g/l) + activated charcoal (1 g/l) + rhizogenic (5 mg/l) supplemented with NAA (0, 0.1, 0.5, 1, 2 mg/l), BA (0, 0.1, 0.5, 1 mg/l). The results of the study (Table 4): After 45 days of culture. The highest regeneration was achieved in culture medium supplemented with BA (0.5 mg/l). The Cymbidium shoots grew well in vitro and have roots in good growth. When the medium only has NAA, PLB formed vigorously and restricted regeneration. In combination with NAA + BA, low regeneration rates and PLB continued to occur. Effects of Tissue and Genotype on Regeneration of callus Effect of tissue culture on regeneration of embryogenic callus PLB was cut into thin slices and young leaves in vitro (10-20 mm) separated shoots were used as culture materials to produce and regenerate the embryonic callus. The culture medium produced the embryonic callus: MS + BA (0.5 mg/l) + peptone (1 g/l). The results of the study showed that (Table  5) the culture medium produced the embryonic callus: MS + BA (0.5 mg/l) + peptone (1 g/l). After 45 days of culturing: Both tissues cultured produced calluses. The culture process was set externally, the embryogenic callus was green; in contrast, in the dark it was ivory-white. The ivory-white embryonic callus cells are suitable for the liquid multiplication of shaking with diffused light. In culture medium, regeneration of embryonic callus: MS + BA (0.5 mg/l) + peptone (1 g/l): After 45 days of culture: callus from both tissues had high shoots, achieving 100% regeneration rate of culture samples. Effects of genotypes on regeneration of embryogenic callus PLB culture specimens from apical shoots and hybrid seed were incubated. Cultured embryogenic callus growth medium: MS + BA (0.1 mg/l) + peptone (1 g/l). The results of the study (Table 6) showed that: After 45 days of culture, both types of PLB were incubated to produce callus. The culture process is set externally, the embryogenic callus was green; in contrast, in the dark it was ivory-white. The ivory-white embryonic callus cells are suitable for the liquid multiplication of shaking with diffused light. The culture medium for regeneration of callus: MS + BA (0.5 mg/l) + peptone (1 g/l) + activated charcoal (1 g/l): After 45 days culture: thin layer of the two samples were cultured to produce high shoots, reach 4-5 shoots/culture and the rate of regeneration of 100% for the samples put into culture.   Source: Author Formation and Proliferation of Callus Suspension Formation of embryogenic callus suspension Callus embryos are used as culture materials to produce a callus suspension. The base liquid medium for callus suspension formation is: MS + BA (0.1 mg/l) + peptone (1 g/l) supplemented with NAA (0.1, 0.5, 1, 3 mg/l), 2.4D (0.1, 0.5, 1, 2 mg/l). The results showed that (Table 7): After 30 culture days, the somatic cell suspension was formed on a suspension culture supplemented with NAA (1 mg/l) or 2.4D (1 mg/l). However, during prolonged culture 2,4D was easy to variate. The appropriate growth regulator in culture was NAA (1 mg/l). The suspension was ivory-white, cells were uniform in shape and size. Source: Author Proliferation of embryogenic callus suspension The somatic cell suspension after 3 times subcultured were used as culture material. The volume of culture was 20%. The culture medium for proliferation of embryogenic callus suspension was: MS + BA cellulose (0.1 mg/l) + peptone (1 g/l) supplemented with NAA (0.1, 0.5, 1, 2, 3 mg/l). The results showed that (Table 8): After 20 culture days, the suspension of somatic cell was rapidly produced on culture medium supplemented with NAA (1 mg/l), with 8-10 times proliferation in biomass. It was ivory-white, cells were uniform in shape and size. Partial somatic cell suspension activity differentiated into somatic embryos.  Source: Author

Regeneration of Embryogenic Callus Suspension Activation induction of somatic embryo
The embryonic callus suspension was used as a culture material. Cell volume of 10 g/50 ml of culture medium induced somatic embryos: MS + peptone (1 g/l) supplemented with BA (1 mg/l). The results of the study show (Table 9): After 45 days of culture, the culture medium of MS + BA (1 mg/l) stimulates somatic embryogenesis. Time of differentiation of embryos increased with time of culture from 1-4 weeks after culture, then gradually decreased. Embryonic induction > 75% (including spherical, heart shape, torpedo), the appropriate time for regeneration. The cells were white-yellow, large, with concentrated cytoplasms.
Splatting and regeneration of somatic embryos on agar medium The embryonic cell suspension was used as a culture material. Cell volume of 5 ml/60 ml of culture medium for regeneration of somatic embryo suspension: MS + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (0.1, 0.5, 1 mg/l) and BA (0.1, 0.5, 1 mg/l). Illumination intensity was 33.3 μmol/m 2 /s. Lighting time was 10 hours/day. Results of the study (Table 10): After 30 days of culture, the somatic embryo culture medium supplemented with BA (1 mg/l) + NAA (0.1 mg/l) was suitable for somatic embryo differentiation. Blue somatic embryos were differentiated into PLB; and shoot regeneration was recorded after 60 culture days with 19 shoo/50ml embryonic cell suspension (Table 4). 3,800 buds of Cymbidium sp. per one liter of embryonic cell suspension were collected.  Source: Author

Conclusion
The PLB was cut in thin slices and used as culture materials. The thin slices were cultured on callus formation medium: MS + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (1 mg/l) or 2.4D (1 mg/l) and proliferation medium: MS + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (1 mg/l) or 2,4D (1 mg/l). The formation and proliferation of callus suspension on medium: MS + peptone (1 g/l) + activated charcoal (1 g/l) supplemented with NAA (1 mg/l) + BA (0.1 mg/l). The medium of regeneration of embryonic callus: MS + peptone (1 g/l) + activated charcoal (1 g/l) + B1 (5 mg/l) + BA (0.5 mg/l). The culture medium to produce embryonic callus: MS + BA (0.5 mg/l) + peptone (1 g/l). After 45 days culturing: Both cultured tissue (PLB sliced and young leaf) produced callus. The culture process was set externally, the embryonic callus was green; in contrast, in the dark it was ivory-white. The ivory-white embryonic callus cells were suitable for the liquid multiplication of shaking with diffused light. In culture medium for regeneration of embryonic callus: MS + BA (0.5 mg/l) + peptone (1 g/l): After 45 days of culture: the callus from both tissues produced shoots, reaching the rate of regeneration of 100% for the samples put into culture. The cultured medium to produce embryonic callus: MS + BA (0.1 mg/l) + pepton (1 g/l) + activated charcoal (1 g/l). After 45 days culturing: Both PLB cultured (from apical shoots and hybrid seeds) produced embryogenic calluses. The ivory-white embryogenic callus cells were suitable for the liquid multiplication of shaking with diffused light. On the culture medium regenerate callus: MS + BA (0.5 mg/l) + peptone (1 g/l): After 45 days of culture: the embryogenic callus from PLB sliced produced high rate shoots, reach 4-5 shoots/culture the rate of regeneration 100% of the samples put into culture. The somatic suspension formed on culture medium: MS + BA (0.1 mg/l) + peptone (1 g/l) + NAA (1 mg/l) or 2.4D (1 mg/l), after 30 days of culture. However, during prolonged culturing, 2.4D is easy to variate. The appropriate growth regulator in culture was NAA (1 mg/l). The suspension was ivorywhite, and cells were of uniform shape and size. The somatic cell suspension regeneration on medium (0.1 mg/l) + peptone (1 g/l) + NAA (1 mg/l) with biomass increments 10.2 times after 20 days cultured. The suspension was ivory-white, and cells were of uniform shape and size. Partial somatic cell suspension activity differentiates into somatic embryos. The cell volume of 10 g/50ml culture medium induced embryos: MS + peptone (1 g/l) + BA (1 mg/l) activation of somatic embryo development after 45 days cultured. Time of differentiation of embryos increased with time of culture from 1-4 weeks after culture, then gradually decreased. Embryogenic induction > 75% (including spherical, heart shape, torpedo), the appropriate time for transplant regeneration. Cell volume of 5 ml/60 ml of culture medium to regenerate somatic suspension: MS + peptone (1 g/l) + BA (1 mg/l) + NAA (0.1 mg/l) suitable for differentiation somatic embryos, forming a somatic embryo cell layer, were blue, they were differentiated into PLB; and shoots regenerated after 60 culture days, 3,800 shoots per one liter of embryogenic cell suspension were collected.