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When the ordinary callus (Fig. 2a, e) was used as the transformation receptor and screened according to the organ regeneration pathway, although the Km concentration increased from the first generation to the fourth generation, the negative (albino buds and white heart buds) rate did not change significantly, at 50%−60% (Table 1). From the 5th generation to the 7th generation, the incidence of green shoots decreased significantly with the increase of Km concentration: to the 7th generation, the incidence of green shoots was only 8.57%, and the elimination rate increased significantly. Since the subculture is to cut the green buds on the previous generation together with the base callus to continue screening, the base of the adventitious buds will continue to differentiate into adventitious buds. The cumulative positive rates were 9.9%, 0.77% and 0.017% after 3, 5 and 7th generation screening, respectively. Using embryogenic callus (Fig. 2b, f) as the receptor material, the cumulative positive rates of the 3rd, 5th and 7th generations were 8.5%, 0.92% and 0.25%, respectively, and the positive rate from the 6th generation was basically stable at about 50%[17].
Table 1. Screening results of pineapple common callus as the transformation receptor.
Number of generations filtered Km
(mg/L)Number of
receptor materialsTotal number of adventitious buds Number of whitening buds Number of white cores Number of green buds Proportion of
green buds (%)1 20 200 357 68 97 192 53.78 2 30 192 358 102 88 168 46.93 3 50 168 383 82 151 150 39.16 4 50 150 197 26 90 81 41.12 5 60 81 251 81 122 48 19.12 6 60 48 84 20 42 22 26.19 7 70 22 35 6 26 3 8.57 Total 0.017 Cumulative green bud rate is the product of green bud rate from each generation. The above results show that receptors obtained from embryogenic callus and the somatic embryo regeneration pathway can significantly improve the screening efficiency. And that the number of screening cultures should be 3−5 generations. Too few screening alternative can increase the difficulty and cost of molecular detection. And too many screening alternatives also reduce screening efficiency.
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The transgenic research of pineapple focuses on establishing a stable and efficient transformation system. The molecular biological identification of the transformed plants is similar to that of other plants. Under the current technical conditions, the pineapple conversion technology system we recommend is shown in Fig. 4. The adventitious bud leaf base produced by tissue culture (Fig. 4a) and undifferentiated calli within three generations are used as receptor materials for infection (Fig. 4b). Then, screening and plant regeneration are performed simultaneously through somatic embryogenesis (Fig. 4, step ②) to increase the transformation frequency and reduce the production of transgenic chimeras. The organogenesis pathway can also be used for simultaneous screening and plant regeneration (Fig.4, step ③). Although the conversion efficiency of this method is slightly lower, the technical difficulty is very low[21,22].
Figure 4.
Flow chart of the pineapple transformation system. (a) Leaf base (Bar = 0.5 cm). (b) Non-embryogenic cells (scanning electron microscopy, SEM, Bar = 20 μm ). (c) Embryogenic cells (SEM, Bar = 50 μm). (d) Somatic embryogenesis, where the red arrows indicate the original embryo (SEM, Bar = 20 μm). (e) Mature somatic embryos (Bar = 500 μm). (f) Adventitious shoot regeneration from callus (Bar = 700 μm). (g) After three consecutive generations of screening, the regenerated plants are green for Km-resistant transformed buds (Bar = 0.5 cm). (h) Somatic embryos produced from embryogenic callus (Bar = 700 μm ). (i) Km-negative plants (Bar = 0.5 cm). (j) Transforming buds (Bar = 0.5 cm). ① Dedifferentiation culture; ② Somatic embryogenesis pathway; ③ Organogenesis pathway; ④ Rooting culture.
Pineapple genetic transformation requires the use of medium and antibiotics. The specific process includes the following steps. First, receptor materials are co-cultured on MS + 3.0 mg/L BA + 2.0 mg/L NAA + 100 μmol/L AS + 8 g/L agar for 3 d. Then they are transferred to selective medium (MS + 3.0 mg/L BA + 2.0 mg/L NAA + 20 mg/L Km + 400 mg/L Carb + 8 g/L agar). This process lasts about 10 d when adventitious buds begin to differentiate. After 28 d, the green Km-resistant adventitious buds are selected and transferred to the second round of screening medium MS + 2.0 mg/L NAA + 30 mg/L Km + 400 mg/L Carb + 8 g/L agar. The third screening and strong seedlings are carried out on the medium MS + 1.0 mg/L NAA + 50 mg/L Km + 400 mg/L Carb + 8 g/L agar. Finally, the green buds are transferred into rooting medium MS + 1.0 mg/L IBA + 50 mg/L Km + 8 g/L agar for culture. It is worth emphasizing that pineapple is a monocotyledonous plant, lacking phenolic substances which are important signalling compounds that induce T-DNA to enter the recipient cells. Therefore, 100 μmol/L Acetosyringone (AS) must be added to the medium during the co-culture stage.
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About this article
Cite this article
He Y, Luan A, Wu J, Zhang W, Lin W. 2023. Overcoming key technical challenges in the genetic transformation of pineapple. Tropical Plants 2:6 doi: 10.48130/TP-2023-0006
Overcoming key technical challenges in the genetic transformation of pineapple
- Received: 03 December 2022
- Accepted: 18 April 2023
- Published online: 29 May 2023
Abstract: In recent years, transgenic technology has become the most important tool for molecular breeding. An efficient genetic transformation system is the key to improving the efficiency of biological breeding, and Agrobacterium-mediated genetic transformation is the common method used in plant genetic transformation experiments. Pineapple is an important tropical horticultural plant, but it has a very narrow genetic base, high genome heterozygosity, and strict self-incompatibility, thus limiting the value of conventional breeding techniques. To shorten the breeding cycle and create new subversive varieties, transgenic research of pineapple is imperative. Due to the characteristics of pineapple, in vitro regeneration technology is relatively straightforward, but it can still be very difficult to obtain pineapple transgenic materials. Over more than 20 years of research on pineapple genetic transformation, we have explored, continuously improved and now established a set of transformation tools for the simple and effective transformation of pineapple genes. The basic premise of our approach is a straightforward redifferentiation of pineapple suckers as explants. Specifically, the receptor material that is the basis for the successful transformation of pineapple is the in vitro culture of callus, which is a tissue that has not yet entered the organ differentiation stage. The nptII gene was selected as the optimal selection marker gene and the somatic embryogenesis pathway is used for screening and regeneration.
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Key words:
- Pineapple /
- genetic transformation /
- explants /
- receptor material /
- regeneration pathways