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Establishment of MMCT in Citrus






MMCT is still an emerging technology for partial genome transfer in plants, with the procedure being completely established only for a few members of the Solanaceae and Compositae families. This technology has a great potential for partial genome


transfer in citrus and especially for the highly polyembryonic species, which are completely dependent on natural or induced mutation for the development of new improved cultivars, as previously dis- cussed. The most important parameter to establish MMCT in citrus is to know the growth rates of suspension cells of several species. According to Verhoeven et al. (1991), a high division activity is very important for the formation of a large number of micronucleated cells, since micronuclei are formed during cell divi- sion. Ramulu et al. (1993), in order to obtain a sustained division activity of sus- pension cells of N. plumbaginifolia, subcul- tured the cells at 3–4 day cycles. A day after subculture, at an early log phase, the cell cycle was synchronized by applying hydroxyurea (HU) or aphidicolin for 24 h, after which, APM was applied. Similar con- ditions were applied to S. tuberosum sus- pension cells (Ramulu et al., 1996b). Binsfeld et al. (2000) observed that on the fourth day of cultivation, suspension cells of H. giganteus and H. maximiliani had their highest mitotic activity and, therefore, cells were treated with APM on the third day, during early log-phase growth without cell cycle synchronization. The conditions used for the induction of high micronucle- ation rates of N. plumbaginifolia and S. tuberosum suspension cells were very suit- able for all citrus species tested and there- fore we have been using a 3–4 day subculture cycle, with cell cycle synchro- nization performed a day after subculture, followed by APM treatment (Louzada, 2001, unpublished data). Cell cycle syn- chronization, 24 h before APM treatment was critical to maintain high micronucle- ation rates in citrus. Concentrations of APM ranging from 24 to 48 mM for 24 h, preceded by treatment with 10 mM HU for 24 h, were very effective in inducing high micronucle- ation rates in ‘Ruby Red’ grapefruit (Citrus paradisi), ‘Valencia’ sweet orange (C. sinen- sis), ‘Changsha’ mandarin (C. reticulata), ‘Murcott’ tangor (C. sinensis ´ C. reticulata), and the citrus relatives Swinglea glutinosa and Microcitrus papuana. The best concen-


 


 

tration, however, for mass production of microprotoplasts was 32 mM APM preceded by 10 mM HU (Louzada, 2002, unpublished data). After this initial APM treatment, pro- toplasts need to be isolated and maintained highly micronucleated. In N. plumbagini- folia (Ramulu et al., 1993) and S. tuberosum (Ramulu et al., 1996b), the presence of APM or cremart plus CB during the entire process of donor protoplast manipulation was required for effi cient fractionation of micronucleated protoplast by ultracentrifu- gation. APM prevented the reformation of MTs in micronucleated protoplasts, and CB disrupted the microfi laments while main- taining the integrity of the plasma mem- brane. Louzada et al. (2002) produced large quantities of microprotoplasts from ‘Ruby Red’ grapefruit and from the citrus relative

S. glutinosa using the procedure described by Ramulu et al. (1993) for N. plumbagini- folia, with a few modifi cations as briefl y described. Early log phase suspension cells (1.0 g fresh weight drained) of ‘Ruby Red’ grapefruit and S. glutinosa were harvested 1 day after subculture (3–4 day cycle) and treated with a freshly prepared solution of 10 mM HU for 24 h. Non-treated suspen- sion cells were used as a control. Treated and control cells were washed four times with H+H medium (Grosser and Gmitter,

 

 

Fig.11.1. A cell with scattered chromosomes.


 

 

Fig.11.2. A cell with several micronuclei.

 

1990) and incubated with 32 mM APM (Bayer Corp., Agricultural Division, Kansas City, Missouri) for 24 h. During this time, cells with scattered chromosomes (Fig. 11.1) of with several micronuclei (Fig. 11.2) could be visualized. After the initial APM treatment, suspension cells were incubated for 24 h in a cell wall digesting mixture containing equal parts of enzyme solution and 0.6 M BH3 medium (Grosser and Gmitter, 1990) supplemented with 32 mM APM, and 20 mM CB (Sigma, St Louis, Missouri). Protoplasts were then fi ltered through a 45 mm stainless steel mesh screen and pelleted at 100. g to remove enzyme. Protoplasts were purifi ed by centrifugation using a 25% sucrose–13% mannitol gradi- ent and washed once with 0.4 M mannitol. During the protoplast manipulations, all solutions contained 32 mM APM and 40 mM CB. A continuous iso-osmotic gradient of percoll was prepared by adding 7.2% (w/v) mannitol to a percoll solution (Amersham Pharmacia Biotech, Piscataway, New Jersey) followed by centrifugation for 30 min at 100, 000. g in a swinging bucket rotor (SW 41 Ti-Beckman Instruments, Inc., Fullerton, California) and 6 ´ 13.2 ml tubes. The protoplast suspension was layered in the pre-formed gradient and centrifuged for 2 h using the above conditions. After cen-


 


 

 

Fig.11.3. A percoll gradient.

 

 

trifugation, bands ‘b’ and ‘c’ (Fig. 11.3) were collected together in 15 ml of 0.6 M BH3 (Grosser and Gmitter, 1990) and fi l- tered sequentially through a 20 mm nylon mesh screen (Small Parts Inc., Miami Lakes, Florida), 14 mm nucleopore mem- brane (Corning, Action, Massa-chusetts) and 10 and 5 mm nylon mesh screens (Small Parts, Inc.). When bands ‘d’ and ‘e’ were dense enough, they were collected together with bands ‘a’ and ‘b’. Filtration was performed by gravity fl ow, and a light pressure was applied, if necessary. Microprotoplasts were collected by two rounds of centrifugation at 80. g and 100. g using a table top centrifuge, and resus- pended in an appropriate volume of BH3 medium (Grosser and Gmitter, 1990). Microprotoplasts were stained with one drop of acridine orange solution at 10 mg/ml and checked for integrity using fl uores- cence microscopy. To determine yield, microprotoplasts were counted under a light microscope using a haemacytometer. In the original protocol described by


Ramulu et al. (1993) and others for plants of the Solanaceae and Compositae families, the microprotoplast bands collected from the percoll gradient were diluted in 0.4 M mannitol, and also used to wash the sieves during the microfi ltration. For citrus, if too much mannitol was collected from the gra- dient, or if the bands were diluted in man- nitol, the microprotoplasts could not be precipitated after fi ltration. Therefore, spe- cial attention must be paid during the removal of bands from the percoll gradient. In cases where more mannitol was col- lected together with the bands, it was very important to dilute further with 0.6 M BH3 medium.

The procedure has been very consis- tent, always producing a large amount of microprotoplasts, of the order of 2 ´ 106 microprotoplasts/g of drained suspension cells, and almost 80% of the microproto- plasts have only one chromosome. A size comparison between protoplast (P) and microprotoplast (M) is shown in Fig 11.4. ‘Ruby Red’ microprotoplasts produced were used in fusion with ‘Succari’ sweet orange protoplasts isolated from embryo- genic suspension cells. No plants were regenerated; however, two embryos were produced. One of the embryos pro- duced roots, enabling us to determine cyto- logically the chromosome number, which was 22.

The swinglea glutinosa microproto-


 


 

plasts were fused with sour orange (C. aurantium) mesophyll protoplasts, but no embryos were obtained, even though the fusion products grew very rapidly and pro- duced microcallus. Suspension cells were produced from the fusion products and many cells containing 2–6 extra chromo- somes were observed. Since mesophyll pro- toplasts from sour orange do not regenerate by themselves, neither do the S. glutinosa microprotoplasts (Louzada, 2001, unpub- lished data), it was expected that many cells would have extra chromosomes. Several other combinations were attempted but, even though callus were obtained, no embryos were produced. Later, we found that the reduction or inhibition of embryo- genesis of the fusion products was directly related to the concentration of CB used during the MMCT process (Louzada, 2001, unpublished data). Lorz et al. (1981) reported a reduction of viability and plating effi ciency of miniprotoplasts using concen- trations of CB from 1 to 200 mg/ml and incu- bation times of up to 24 h. By reducing the concentration of CB from 20 to 10 mM during the enzyme incubation, and from 40 to 10 mM during the percoll gradient, we were able to produce a large amount of embryos from several fusion combinations. The majority of the embryos were bipolar and produced roots and shoots; however, the roots were initially very vigorous, but when the shoots were approximately 1.5 cm, they reduced their growth rates dramat- ically. Attempts to produce adventitious roots failed in all cases, therefore, the shoots were micro-grafted in vivo (Skaria, 2000). Cytology performed in root tips of several plantlets from the fusion ‘Succari’ sweet orange (recipient) + ‘Ruby Red’ grapefruit (donor) revealed chromosome numbers ranging from 2 n + 1 to 2 n + 6. This fusion combination was cultured in EME medium (Grosser and Gmitter, 1990) which contains 0.6 M sucrose. It is well known that the embryogenesis capacity of sweet orange is inhibited in medium with a sucrose concentration higher than 0.3 M (Ohgawara et al., 1985), and the micro- protoplasts do not divide when cultured


 

 

Fig.11.5. A micro-grafted microprotoplast hybrid.

 

 

alone (Louzada, 2001, unpublished data). We therefore, expected that only those with extra chromosomes would be able to regenerate into plants. All cyto- logical analyses performed so far in root tips revealed additional chromosomes. A micro-grafted plant is shown in Fig. 11.5, and a root tip cell containing 24 chromo- somes (2 n + 6) is shown in Fig. 11.6. We are currently micro-grafting plantlets from sev- eral other fusion combinations. Molecular characterization of all the plants is under- way.

 

 


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