The different hormones have effect on leaf callus induction with the frequency and size of callus formation as the main index and the color and quality of callus as a reference. Finally, the results have revealed that the best medium component about leaves of poinsettia inducing callus formation contain MS, NAA 1.0 mg/L, 6-BA 0.1mg/L, sucrose 30 mg/L, agar 6.0 g/L and its PH value is 5.8 (Fig 1a; Table 4). The different hormones have influence on callus inducing cluster buds formation with the germination rate as the main index and the quantity and length of buds and the better growth vigor as references, which have indicated that the best medium component of hormones about leaf callus inducing cluster buds formation comprise 6-BA 1.0 mg/L, KT 0.4 mg/L, NAA 0.1 mg/L and 2,4-D 0.05 mg/L. The different 6-BA concentration and the different kind of auxin have impact on the cluster buds proliferation with proliferation rate as the main index, referencing the quantity and length of effective buds and better growth vigor. In conclusion, the medium component about leaf callus inducing cluster buds proliferation include 6-BA 1.2 mg/L and NAA 0.05 mg/L (Fig 1b; Table 5). The fractional culture of different budling and Ac addition have effect on strong seedling cultivation of cluster buds (Firoozabady and Gutterson, 2003)
. The different hormones have influence on rooting with length of the root and quantity of the first level root and the second level root deemed to the main index, referencing the height of seedling and the better growth vigor. Lastly, the medium component about leaf inducing cluster buds to take root contain IBA 0.8 mg/L, NAA 0.5 mg/L and 6-BA 0.1 mg/L (Fig 1c; Table 6).
Table 4: Multiple comparison of callus inducting cluster buds between combinations affected by the different hormone.
Table 5: Multiple comparison of cluster buds reproduction between combinations.
Stems with buds regeneration
Table 6: Multiple comparison of different hormones impacting rooting between combinations.
The different concentrations of 6-BA and NAA have influence on inducing cluster buds of stem with bud to germinate (Jiao et al.
, 2017). The medium component about stem with bud inducing cluster buds to germinate comprise 6-BA 1.5 mg/L and NAA 0.1 mg/L (Fig 1d). The different 6-BA concentration and the different kind of auxin have effect on the cluster buds proliferation, which the medium component inducing cluster buds proliferation include 6-BA 1.0 mg/L and NAA 0.05 mg/L (Fig 1e; Table 5). The different addition level of Ac and fractional culture of different budling have influence on strong seedling cultivation of cluster buds. The different hormones and different addition level of Ac have effect on rooting with the length of the root and quantity of the first level root and the second level root deemed to the main index, and the height of seedling and the better growth vigor as references. Lastly, the results have showed that the medium component inducing cluster buds to take root include IBA 1.2 mg/L, 6-BA 0.1 mg/L, Ac 200 mg/L (Fig 1f; Table 7).
Table 7: Multiple comparison of the different hormones and addition level of activated carbon having influence on rooting between combinations.
Fig 1: Establishment of poinsettia leaf and stem with buds regeneration (a) Leaf callus induction; (b) Cluster buds proliferation; (c) Callus rooting at the base of cluster buds; (d) Bud enlargement; (e) Bud germination and vigorous growth; (f) Clustering callus rooting at bud base.
The tender leaves which selected as ex-plants can easily induce callus (Grotkass et al., 1995).
This experiment found that the callus induction rate of the leaves at the top of the shoots was high and the callus induction time was about 7 days earlier than that of other leaves. The callus was induced after being cut on the leaves of the Poinsettia, and was easy to grow from the wound with the strong differentiation (Wang et al., 2013; Jantasorn et al., 2021).
This experiment found that cutting 2 horizontally along the middle of the main vein of the leaf has a higher rate of producing callus than other parts of the wound. The callus was pale yellow and granular. This callus was more prone to dedifferentiation to produce cluster buds.
In the process of multiplication and cultivation of cluster buds, the appropriate addition of Ac can inhibit the proliferation of a large number of weak buds and play a role in seedlings (Jiao 2001)
. Combining the proliferation of cluster buds and the amount of Ac, it can not only save the cultivation time, but also reduce the investment and achieve the purpose of reducing costs and increasing efficiency (Mali and Chavan, 2016)
. In the further study, as the Poinsettia leaves induce the cluster buds to conduct outdoor hydroponic rooting, it is hoped that this will simplify the tissue culture procedure, reduce the indoor culture time and production costs, and enhance the ability of the tissue culture seedlings, which adapt to the external environment and improve the survival rate of tissue cultured seedlings (Cheng et al., 2016
; Thakuria et al., 2018
Poinsettia shoots with buds induce cluster buds. If the culture time is too long, the ex-plants and the medium are prone to produce secretions. And the secretions affect the differentiation, proliferation and growth of the cluster buds (Huang et al., 2014; Kenasa et al., 2021).
This experiment found that in the process of cultivating shoots with buds, especially during the transfer process, the clustering buds were easy to flow out of white jelly from the wound after being cut. If the jelly has been absorbed in time, it was easy to cause endophytic infection, which affected the growth of sterile seedlings.
The appropriate addition of Ac can inhibit the proliferation of a large number of weak buds and overcome the growth of endophytic bacteria and adsorb substances that inhibit the growth of seedlings (Moustaka et al., 2018)
. This is the first report of an efficient plant regeneration protocol for poinsettia through direct organogenesis using ex-plants. Hence, this provides a more efficient basis for the genetic transformation than previously available (Moustaka et al., 2020
; Trang et al., 2021