# GreenTray+
## 4 Discussion  
The extraction of shoots with forceps from in vitro culture containers, as well as from TIS bioreactors, followed by arranging them on sterile surfaces in order to divide them with the help of surgical scalpels and their subsequent introduction into new culture containers, a process known as subculturing in the conventional in vitro culture process, is time-consuming and represents a significant portion the hand labor involved in most in vitro plant culture processes. The components of the actual GreenTray(r) TIS bioreactor described herein, which is a further evolution of the ones presented in the past [31; 32], make it significantly improved and versatile compared to other commercial TIS bioreactors, such as RITA(r), Matis(r), Plantform(tm), SETISTM[1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17], or the TIS developed by several in vitro plant culture laboratories oriented to solve particular propagation objectives for diverse plant species [18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28]. GreenTray provides the opportunity to automate the in vitro clonal plant propagation process because of its new methods: the disposition of the plant material, extraction from the culture vessel, easy manipulation for reorientation, shoot cutting, and reintroduction for the further growth of the shoot basis. These facts represent a significant reduction in the time required for conventional subcultures, implying a reduction in hand labor and an increase in the number of shoots produced per container, which reduce the cost per plant produced and therefore facilitates the application of micropropagation to more plant species. Although excellent shoot development and sequential shoots cuts were obtained for Rootpac(r) R, Rootpac(r) 20, GF677, Intensia(r), and Ferimon, further research is needed to improve the culture media, the frequencies of immersion, and aeration for Garnem(r), Adara, OHF 87 TM, and Py170. Indeed, this observation is applicable to all plant species for which a great amount of experience has been accumulated during previous decades of work with semisolid culture media, compared to TIS culture systems. Figure 13: Levels of affectation by _Erwinia amylovora_ on three apple varieties (**A**) tested for tolerance in the GreenTray®, and (**B**) their correlation with the known effect levels observed with potted plants. Mean values come from the measures of 15 replicates per genotype. Bars represent the standard error. Means with different letters (a, b) are significantly different (Tukey–Kramer HSD, \(p<0.05\)). As reported in previous works [41; 42; 43; 44; 45; 46], autotrophic in vitro culture is facilitated by aeration in the culture vessel and increasing the PAR radiation without stressing the plant materials. GreenTray(r)'s design is particularly focused on improving aeration through two mechanisms, suction from the glass bottle containing the medium or the propulsion of air into the glass culture jar. PAR radiation was significantly increased with LED lights, with a variable and high range of intensities, paired with low heating effects. Aeration of the culture flask induces transpiration in the plantlet's leaves; therefore, water exits through the open stomas and CO\({}_{2}\) diffuses in, which, in return, favors photosynthesis [41]. Rooted plantlets of the _Prunus_ rootstocks Rootpac(r)20 and Garnem(r), or the apple varieties Carrandona, Florina, and Rozona, grew in the GreenTray(r) without sucrose in the culture medium during the biotic and abiotic stress agent assays that lasted several weeks. As an example of a long-term in vitro autotrophic culture, the _Kalandchoe dagrrenontiana_ species demonstrated that the autotrophic culture conditions in the GreenTray(r) are feasible for at least one year. GreenTray(r)'s design allows access to shoots or plantlets by moving the container's lid and attached tray sideways.