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Culture in vitro of grafted plants






Micrografted plants are cultured in a liquid nutrient medium composed of the plant cell culture salt solution of Murashige and Skoog (1962), modifi ed White’s vitamins and 75 g/l sucrose (Navarro et al., 1975). The medium is distributed into 25 ´ 150 mm test tubes in 25 ml aliquots. A folded paper platform, perforated at its centre for insertion of the root portion of the root- stock, is placed in the nutrient solution (Fig. 17.1j). The cultures are kept at con- stant 27°C and exposed 16 h daily to 40–50

mE m–2 s–1 illumination.

Several experiments have been done using auxins and cytokinins in the culture medium or as applications at the grafting point to increase the incidence of grafting success (Navarro et al., 1975; Edriss and Burger, 1984; Starrantino et al., 1986). The treatments gave either negative results or


 

some small increase in grafting success, not confi rmed in other laboratories. In practice, no growth regulators are used for STG.

Sucrose concentration in the nutrient medium of grafted plants has an important infl uence on grafting success and growth of plants (Navarro et al., 1975). The highest rate of successful grafts was obtained with 75 g/l of sucrose. There was also a progres- sive increase in the number and size of new leaves arising from shoot-tips of successful grafts and in the production of new lateral roots by the rootstock with increasing sucrose concentration up to 75 and 100 g/l. Histological studies showed that three days after grafting there was already some callus development between the rootstock and the shoot-tip, and fi ve days after graft- ing the callus was completely developed at the graft union (Fig. 17.1k). Initiation of vascular differentiation was observed seven days after grafting, and there was a com- plete vascular connection between the shoot-tip and the rootstock 11 days after

grafting (Li Xiancai, 1997).

Growing small leaves from the shoot- tip coming out from the incision can be observed about 3–4 weeks after grafting (Fig. 17.1l), and 4–6 weeks after grafting the successful grafts already have 2–4 expanded leaves and they can be trans- planted to soil (Fig. 17.1m).

In the Citrus Variety Improvement Programme in Spain (CVIPS), carried out in our laboratory, this standard procedure has been used with many genotypes of at least 43 Citrus species, and with Poncirus and Fortunella species. The overall percentage of grafting success has been 38%, but dif- ferences were found between species and shoot-tip sources (Table 17.1). Comparative studies using the same genotype have not been done, but shoot-tips excised from bud- wood cultured in vitro gave a slightly higher incidence of grafting success than shoot-tips from plants growing in a growth chamber, albeit that this is species depend- ent. In the case of satsumas the grafting suc- cess has been much higher with shoot-tips isolated from budwood cultured in vitro (Table 17.1). Overall, more than 40% graft-


 

Table 17.1. Average grafting success (%) of shoot-tip grafting in vitro carried out in the Citrus Variety Improvement Program in Spain between 1985 and 2003.

 

Group of genotypes Shoot-tips excised from plants growing in growth chambers Shoot-tips excised from budwood cultured in vitro
All genotypes 33.6 43.5
Sweet oranges    
Clementines   48.2
Satsumas 18.1 39.8
Other mandarins 39.3 45.8
Lemons 19.4 21.2
Grapefruit 58.3

 


ing success was usually obtained with most commercially grown species using bud- wood cultured in vitro as the source of shoot-tips, with the exception of lemons that gave only about 20% success.

 

 

Transplanting to soil

Scions of successful grafts should have at least two expanded leaves before being transplanted to soil. This stage is usually reached 4–6 weeks after grafting (Fig. 17.1n). Micrografted plants are transferred to pots containing a steam-sterilized artifi - cial soil mix suitable to grow citrus. Pots are enclosed in polyethylene bags that are closed with rubber bands and placed in a shaded area of a temperature-controlled greenhouse set at 18–25°C. After 8–10 days, the bags are opened, and after another 8–10 days, the bags are removed and the plants are grown under regular greenhouse condi- tions (Fig. 17.1l). Alternatively, the epicotyl of the micrografted plants can be regrafted on vigorous seedlings growing in the green- house (De Lange, 1978). With both proce- dures, we usually obtain over 95% survival, but growth is faster with the latter method. Plants recovered by STG do not have juvenile characters, if the shoot-tips are excised from adult plants. They usually fl ower and set fruits within two years from grafting (Fig. 17.1l). Several thousands plants have been obtained by STG in differ- ent laboratories, and all available data indi-

cate that they are true to type.







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